Hdac inhibitors and hormone targeted drugs for the treatment of cancer

ABSTRACT

The present invention relates to the methods of treating endocrine-regulated cancers, including hormone resistant cancers, for example. More specifically, the present invention relates to a method of increasing the sensitivity of hormone resistant cancers to hormonal therapeutic agents. In particular embodiments, the present invention concerns delivery of a histone deacetylase inhibitor and a hormone targeted drug to an individual with cancer. In specific embodiments, the histone deacetylase inhibitor and the hormone targeted drug act synergistically to treat the cancer, including by overcoming resistance to a cancer therapy.

This application claims priority to U.S. Nonprovisional patentapplication Ser. No. 12/134,717, filed Jun. 6, 2008, and to U.S.Provisional Patent Application Ser. No. 60/942,452, filed Jun. 6, 2007,and to U.S. Provisional Patent Application Ser. No. 61/013,570, filedDec. 13, 2007, all of which applications are incorporated by referenceherein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with government support under Grant Nos.R01 CA-62483 and R21 CA117991 awarded by the National Institutes ofHealth. The United States Government has certain rights in theinvention.

TECHNICAL FIELD

The present invention relates at least to the fields of medicine andoncology. More specifically, the present invention relates to methodsand compositions of treating cancers, including hormone-resistantcancers, for example. In addition to treating the exemplaryhormone-resistant cancers, the present invention can be used to increasethe sensitivity of cancers to therapy, such as increasing thesensitivity of hormone-resistant cancers to hormonal therapeutic agents,by administering a combination of one or more histone deacetylaseinhibitors and one or more hormonal therapeutic agents.

BACKGROUND OF THE INVENTION

Hormone resistance is a particular problem in cancers such as prostatecancer and breast cancer.

Androgens, acting via androgen receptors, are essential for normalgrowth and function of the prostate gland and have been implicated inthe progression of prostate cancer. Selective androgen receptormodulators (SARMs)—drugs intended to inhibit the activity of androgenreceptors—are therefore standard treatment for prostate cancer. However,prostate cancers often become resistant to such treatment. A similarphenomenon can also occur in breast cancers treated with drugs thattarget the hormone receptor for estrogen.

A. Breast Cancer

Breast cancer is the most prevalent form of cancer among women in theUnited States and second leading cause of cancer related deaths (Jemalet al., 2006). According to 2006 cancer statistics, approximately 40,000women are expected to die from breast cancer in the US (Jemal et al.,2006). In the year 2003, although a marked 7% decrease in the incidenceof breast cancer was reported, this decrease mainly was associated withestrogen receptor positive (ER+) breast cancers (Ravdin et al., 2006).Estrogen receptor-negative (ER−) breast cancer still is essentiallyincurable and aggressive. Although, breast cancer treatment hasundergone significant improvement, resistance develops to almost allforms of cancer therapy. Additionally, there has been little improvementin the treatment of ER− breast cancer. The high prevalence of breastcancer and development of resistance to effective treatments provides astrong stimulus for the development of additional, targeted therapieswith minimal toxicity.

Hormone resistance is a particular problem in cancers such as breastcancer. The antiestrogen tamoxifen, intended to inhibit the activity ofestrogen receptor, is standard treatment for breast cancer. However,breast cancers often become resistant to such treatment.

The knowledge that estrogens contribute a pivotal role in development ofbreast cancer has been exploited clinically by the development ofendocrine agents, predominantly by estrogen withdrawal or antagonism(Jordan et al., 2007). Antiestrogen tamoxifen has been used to treatbreast cancer for a number of years now. More recently, AIs such asletrozole and anastrozole have surpassed the beneficial effects oftamoxifen and are now being used in the clinic as the first linetreatment for hormone dependent post-menopausal breast cancer (Goss etal., 2005; Goss et al., 2002). AEs/AIs are currently used forpostmenopausal ER positive breast cancer agents (Brodie, 1990; Baum,2002; Baum et al., 2002). The clinical use of these agents is limited bydevelopment of resistance and the presence of ER− cancer phenotype. Lossof AE/AI sensitivity has been associated with lack of ER expression.

B. Prostate Cancer

Prostate cancer is the most commonly diagnosed cancer in North Americanmen and it is estimated that there are over 300,000 newly diagnosedcases each year (Landis, et al., 1998; Shibata, et al., 1998). Theincidence and mortality rates from prostate cancer are increasing andthis is due, in part, to an increasingly aging population and the higherincidence of this disease in older men (Gao et al., 1997; Chiarodo,1991). Both benign prostatic hypertrophy (BPH) and prostate cancer aredecreased or not detected in eunuchs and are linked not only toadvancing age but the presence of testes and androgen function (Gao etal., 1997; Chiarodo, 1991; Sakti and Crawford, 1993).

Early prostate cancer tends to be androgen-dependent and requiresexpression of a functional androgen receptor (AR), whereas later stagetumors progress to androgen-independence which in some cases iscorrelated with loss of AR function (Cheng et al., 1993). Interestingly,the progression from early stage hormone-dependent to latter stagehormone-independence in prostate cancer in men is also observed forbreast cancer in women where estrogen-responsiveness undergoes a similarpattern of change in women with early or late stage disease (Hopp andFuqua, 1999; Fuqua et al., 1995).

Prostate cancer therapy is dependent on the stage of the tumor and ARexpression. Early stage androgen-responsive prostate cancers can betreated by castration or with antiandrogens or drugs that blockandrogen-induced responses including steroidal antiandrogens(cyproterone), LHRH analogs, nonsteroidal antiandrogens (flutamide,nilutamide, bicalutamide), and the potent estrogenic drugdiethylstilbestrol (reviewed in (Sadar et al., 1999; Klotz, 2000; Morriset al., 2000; Boccardo, 2000). In addition, there are several possiblenovel strategies for treatment of prostate cancer and other tumor-typesand these include targeting of critical genes involved in tumor cellgrowth and metastasis (e.g., antiangiogenic drugs, antisense therapy)(Boasberg et al., 1997; Knox et al., 1998; 1998; Yamaoka et al., 1993;Folkman, 1995; Folkman, 1971). Ligands for nuclear receptors (NR) arealso being developed for treatment of prostate cancer through inhibitoryNR-AR crosstalk that involves various ligands or drugs that bind theretinoid acid/X-receptors (retinoids), vitamin D receptor (calcitrol),and peroxisome proliferator activate receptor γ (trogilatazone) (Doraiet al., 1997; Pienta et al., 1993; Pollard et al., 1991; Kelly et al.,1996; Miller et al., 1992; Miller et al., 1995; Peehl et al., 1994;Gross et al., 1998; Kubota et al., 1998; Tontonoz et al., 1997; Tontonozet al., 1994; Smith et al., 1999).

The present invention is the first to use a combination of hormonaltherapy and histone deacetylase inhibitors to treat hormone resistantcancers.

SUMMARY OF THE INVENTION

In particular cases, the present invention concerns treatingendocrine-regulated cancers, including, by way of non-limiting example,breast, prostate, ovarian and endometrial cancers. In specificembodiments, the endocrine-regulated cancer is resistant to one or moretherapies, whereas in other embodiments the cancer is sensitive to oneor more therapies. In further specific embodiments, theendocrine-regulated cancer is resistant to one or more endocrinetherapies, whereas in other embodiments the cancer is sensitive to oneor more endocrine therapies. In particular embodiments, the cancer ishormone-resistant cancer, such as estrogen-resistant cancer orprogesterone-resistant cancer, for example.

In certain aspects of the invention, there are methods and compositionsthat overcome resistance to a cancer therapy, including an endocrinetherapy, using histone deacetylase inhibitors (HDACi) in combinationwith a hormone-targeted drug. In additional aspects, the presentinvention concerns enhancing response of a cancer to therapeutic agents,and in specific aspects the present invention concerns enhancingresponse of a cancer to endocrine agents. In further aspects, thepresent invention concerns synergism between HDACi and hormone-targeteddrugs for the treatment of cancer, and in particular for the treatmentof a cancer that is endocrine regulated, including one that is resistantto the corresponding endocrine hormone.

The present invention relates to the use of HDACi to alter thesensitivity of estrogen receptor negative cells or androgen receptornegative cells to hormonal therapies. For example, the HDACi increase ERand aromatase expression and/or activity, thus making the tumors orneoplasms more sensitive to aromatase inhibitors in addition toantiestrogens. In some embodiments, the combination of HDACi andhormonal therapies have an additive or synergistic inhibitory effect onhormone dependent cells and tumors.

One embodiment of the present invention comprises a method of treating ahormone resistant cancer comprising the step of administering to asubject having or suspected of having the hormone resistant cancer ahistone deacetylase inhibitor and a hormone targeted drug in aneffective amount to treat the cancer. The hormone resistant cancer isbreast cancer, in a specific embodiment. More specifically, the breastcancer is estrogen receptor negative. Still further, the breast cancercan be refractory.

In certain preferred embodiments, the hormone therapy comprises anaromatase inhibitor or an anti-androgen.

In certain embodiments, the hormone targeted drug is an aromataseinhibitor. Aromatase inhibitors include compounds that inhibit theaction of the enzyme aromatase, which converts androgens into estrogensby aromatization. Aromatase inhibition by a particular compound can bedetermined by using methods known in the art including measuring therelease of tritium-labeled water upon the conversion of tritium-labeledandrostenedione to estrone in the manner provided for in U.S. Pat. No.6,803,206, which is incorporated herein by reference in its entirety.

Exemplary aromatase inhibitors useful in the invention can include, butare not limited to, anastrozole (Arimidex), letrozole (Femara),exemestane (Aromasin), formestane (Lentaron), and testolactone (Teslac).Other compounds that have shown promise as aromatase inhibitors that maybe used in the present invention include, but are not limited toatamestane, vorozole (Rivizor), fadrozole (16949A), roglethimide,pyridoglutethimide, trilostane, aminoglutethimide (Cytadren),4-Hydroxyandrostenedione (4-OHA; Formastane), finrozole, and YM-511(4-[N-(4-bromobenzyl)-N-(4-cyanophenyl)amino]-4H-1,2,4-triazole).

In certain embodiments, the hormone targeted drug is an anti-androgen.As used herein, the term “anti-androgen” refers to a compound that thatcan prevent or inhibit the biologic effects of androgens inhibitingactivity of the C17,20-lyase or 5-reductase enzymes. In specificembodiments, the term comprises a compound that inhibits binding betweenandrogen and an androgen receptor. As used herein, a17-alpha-hydroxylase-C17,20-lyase inhibitor is a compound that directlyor indirectly inhibits the activity of either one or both of the enzymesC17,20-lyase or 5-reductase, which converts testosterone (T) to5-alpha-dihydrotestosterone. Examples of hydroxylase/lyase and/or5-reductase inhibitors can be found in U.S. Pat. No. 5,994,334; U.S.Pat. No. 5,264,427; and WO 2006/093993. Compounds that inhibit theandrogen receptor can be identified using methods known in the artincluding one or more in vitro cell-assays that profile ligand mediatedmodulation of the androgen receptor, including, but not limited to (i)N—C interaction, (ii) transcriptional repression, and (iii)transcriptional activation, as set forth in U.S. Publ. No. 2008/0125399,which is incorporated herein by reference. Inhibition of C17,20-lyaseactivity can be determined by methods known in the art includingmeasuring the release of tritium-labeled acetic acid during theconversion of tritium-labeled hydroxypregnenolone todehydroepiandrosterone using liquid scintillation spectrometry in themanner provided for by Nnane et al which is incorporated herein byreference in its entirety. See Inhibition of Androgen Synthesis in HumanTesticular and Prostatic Microsomes and in Male Rats by Novel SteroidalCompounds, Endocrinology, Vol. 140, No. 6. Similarly, Nnane et al. alsoprovide for an assay for measuring the activity of 5-reductase bycalculating the percentage conversion of tritium-labeled Testosterone(T) to tritium-labeled dihydrotestosterone (DHT) using liquidscintillation spectrometry.

Specific non-limiting examples of anti-androgens useful in the presentinvention include, but are not limited to, spironolactone (Aldactone,Spiritone; Novo-Spiroton, Spiractin, Verospiron or Berlactone),cyproterone acetate (Androcur, Climen, Diane 35, Ginette 35), flutamide(Eulexin), nilutamide (Anandron, Nilandron), bicalutamide (Casodex)which can be formulated, e.g. as disclosed in U.S. Pat. No. 4,636,505,which is hereby incorporated by reference. Other anti-androgen agentsthat may be used in the present invention include, but are not limitedto, those agents described in WO 02/03912, which is incorporated hereinby reference.

The histone deacetylase inhibitor (HDACi) can include, but are notlimited to, those HDACi inhibitors described throughout the application.In some embodiments, the HDACi is SAHA, CI-994, MS-275,3-(1-Methyl-4-phenylacetyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamide(APHA), apicidin, sodium butyrate, (−)-depudecin, scriptaid, sirtinol,trichostatin A and a combination thereof.

In some embodiments, the HDACi is a selective HDACi. For example, inspecific embodiments, the Class I selective HDAC inhibitor is, by way ofnon-limiting example, MGCD-0103(N-(2-amino-phenyl)-4-[(4-pyridin-3-yl-pyrimidin-2-ylamino)-methyl]-benzamide),MS-275 (N-(2-aminophenyl)-4-(N-(pyridin-3-ylmethoxycarbonyl)aminomethyl)benzamide, SNDX-275), spiruchostatin A, SK7041, CI-994, SK7068 or6-amino nicotinamides.

In some embodiments, the HDACi is a non-selective HDAC inhibitor. Inspecific embodiments, the non-selective HDAC inhibitor is, by way ofnon-limiting example, N′-hydroxy-N-phenyl-octanediamide (suberoylanilidehydroxamic acid, SAHA), pyroxamide, CBHA, trichostatin A (TSA),trichostatin C, salicylihydroxamic acid (SBHA), azelaic bihydroxamicacid (ABHA), azelaic-1-hydroxamate-9-analide (AAHA), depsipeptide,FK228, 6-(3-chlorophenylureido) carpoic hydroxamic acid (3Cl-UCHA),oxamflatin, A-161906, scriptaid, PXD-101, LAQ-824, CHAP, MW2796, LBH589or MW2996.

In some preferred embodiments of the invention, the HDACi is selectedfrom one of the following groups or is a specific compound identified inone of the following groups: The short-chain fatty acids (e.g., butyrateand phenylbutyrate, isovalerate, valproate, 4-phenyl butyrate (4-PBA),phenylbutyrate propionate, butyaramide, isobutyaramide, phenylacetate,3-bromopropionate, tributyrin, valproic acid, and Pivanex); hydroxamicacids (e.g., the trichostatins such as TSA and TSC, suberoylanilidehydroxamic acid (“SAHA”) and its derivatives, Oxamflatin, azelaicbiyhydroxamic acid (“ABHA”), azelaic-1-hydroxamate-9-anilide (“AAHA”),suberoyl bishydroxamic acid (“SBHA”), m-carboxycinnamic acidbishydroxamide (“CBHA”), pyrozamide, salicylbishyudoxamic acid,Scriptaid, Pyroxamide, Propenamides, LBH589, CHAP, MY29996, MW2976, andany of the hydroximic acids disclosed in U.S. Pat. Nos. 5,369,108;5,932,616; 5,5,700,811; 6,087,367; and 6,511,990);epoxyketone-containing cyclic tetrapeptides (e.g., trapoxins,depeudecin, depsipeptide FK228, FR 225497, Apicidin, cyclictetrapeptide, Apicidin Ia, Apicidin Ib, Apicidin Ic, Apicidin IIa,Apicidin IIb, a cycli tetrapeptide containing a2-amino-8-oxo-9,10-epoxy-decanoyl moiety, a cyclic peptide without the2-amino-8-oxo-9, 10 epoxy-decanoyl moity, HC-toxin, Chlamydocin,Diheteropeptin, WF-3161, Cyl-1 and Cyl-2); non-epoxyketone-containingcyclic tetrapeptides (e.g., FR901228, Apicidin,cyclic-hydroxamic-acid-containing peptides (CHAPs); benzamides (e.g.,MS-275 (MS-27-275), N-acetyldinaline, CI-994, MGCD0103, other benzamideanalogs); and other miscellaneous structures (e.g., Savicol, Bacecca,MG98, Depudecin, Organosulfur compounds).

In certain embodiments of the present invention, the histone deacetylaseinhibitor and the hormone targeted drug are administered simultaneouslyor the histone deacetylase inhibitor is administered prior to theadministration of the hormone targeted drug.

Another embodiment of the present invention comprises a method ofincreasing sensitivity of a cancer cell to a hormone therapy comprisingthe step of administering to the cell a histone deacetylase inhibitor inan effective amount to increase the sensitivity of the cancer cell. Morespecifically, the cancer cell is a breast cancer cell. The cancer cellscan be comprised within a subject in need of treatment thereof.

Still further, the breast cancer cell is estrogen receptor negative andadministration of the histone deacetylase inhibitor increases theexpression of estrogen receptors in the estrogen receptor negativebreast cancer cell, thereby treating the breast cancer. Alternatively,the HDACi increase aromatase expression and/or activity, thus making thetumors or neoplasms more sensitive to aromatase inhibitors in additionto antiestrogens.

In certain embodiments, the prostate cancer cell is androgen receptornegative and the histone deacetylase inhibitor increases the expressionof androgen receptors on the androgen receptor negative prostate cancercell.

Another embodiment of the present invention comprises a method ofinhibiting growth of a cell comprising contacting the cell with anaromatase inhibitor and a histone deacetylase inhibitor, each in aneffective amount to inhibit growth of the cell. The cell is a cancercell, more specifically, the cancer cell is refractory. Still further,the cancer cell is estrogen receptor negative. In certain embodiments,the histone deacetylase inhibitor increases the expression of anestrogen receptor on the cell.

In a further embodiment, the contacting step occurs in vitro. Stillfurther, the contacting step can occur in vivo. More specifically, thecontacting step occurs in a subject in need thereof.

In particular embodiments, there is a method of inhibiting growth of acell comprising contacting the cell with an aromatase inhibitor and ahistone deacetylase inhibitor, each in an effective amount to inhibitgrowth of the cell, such as a cancer cell. The cancer cell may berefractory, in certain embodiments. In specific embodiments, the cancercell is estrogen receptor negative or progesterone receptor negative,for example. In some cases, the histone deacetylase inhibitor increasesthe expression of an estrogen receptor on the cell. The contacting ofmethods of the invention may be in vitro or in vivo, and it may occur ina subject in need thereof.

In certain embodiments, there is a method of treating anendocrine-regulated cancer in an individual, comprising administering toan individual in need thereof a therapeutically effective amount of ahistone deacetylase inhibitor and a therapeutically effective amount ofa hormone targeted drug. In a specific embodiment, the cancer has aresistance to treatment with the hormone targeted drug when the hormonetargeted drug is administered in an endocrine therapy that does notinclude the administration of a histone deacetylase inhibitor. Theresistance may be de novo resistance or acquired resistance, inparticular embodiments. In some cases, the cancer responds to treatmentwith the hormone targeted drug when administered in an endocrine therapythat does not include the administration of a histone deacetylaseinhibitor.

In specific embodiments, the histone deacetylase inhibitor and thehormone targeted drug act on the cancer synergistically, and/or thehistone deacetylase inhibitor and the hormone targeted drug areadministered simultaneously or sequentially. For example, in some cases,the histone deacetylase inhibitor and the hormone targeted drug areadministered sequentially and the histone deacetylase inhibitor isadministered prior to the hormone targeted drug.

In particular embodiments, the endocrine-regulated cancer is breastcancer, prostate cancer, ovarian cancer, or endometrial cancer. Thecancer may be estrogen receptor negative, progesterone receptornegative, androgen receptor positive, or androgen receptor negative, incertain aspects. The cancer may be positive or negative for aromataseexpression. Furthermore, the hormone targeted drug is an aromataseinhibitor, such as anastrozole, exemestane, letrozole, or combinationsthereof, in certain aspects.

In other embodiments, there is a method of treating anendocrine-regulated cancer in an individual, comprising administering atherapeutically effective amount of a histone deacetylase inhibitor anda therapeutically effective amount of a hormone targeted drug to theindividual, wherein the method overcomes resistance in theendocrine-regulated cancer to the hormone targeted drug. The resistancein the endocrine-regulated cancer may be de novo resistance or acquiredresistance, in certain aspects. The histone deacetylase inhibitor andthe hormone targeted drug are administered simultaneously orsequentially, in some embodiments.

In a specific embodiment, the cancer has a resistance to treatment withthe hormone targeted drug when the hormone targeted drug is administeredin an endocrine therapy that does not include the administration of ahistone deacetylase inhibitor. The resistance may be de novo resistanceor acquired resistance, in particular embodiments. In some cases, thecancer responds to treatment with the hormone targeted drug whenadministered in an endocrine therapy that does not include theadministration of a histone deacetylase inhibitor.

In specific embodiments, the histone deacetylase inhibitor and thehormone targeted drug act on the cancer synergistically, and/or thehistone deacetylase inhibitor and the hormone targeted drug areadministered simultaneously or sequentially. For example, in some cases,the histone deacetylase inhibitor and the hormone targeted drug areadministered sequentially and the histone deacetylase inhibitor isadministered prior to the hormone targeted drug.

In particular embodiments, the endocrine-regulated cancer is breastcancer, prostate cancer, ovarian cancer, or endometrial cancer. Thecancer may be estrogen receptor negative, progesterone receptornegative, androgen receptor positive, or androgen receptor negative, incertain aspects. Furthermore, the hormone targeted drug is an aromataseinhibitor, such as anastrozole, exemestane, letrozole, or combinationsthereof, in certain aspects.

In other embodiments, there is a method of enhancing a response of anendocrine-regulated cancer in an individual to a hormone targeted drug,comprising administering to an individual in need thereof atherapeutically effective amount of a histone deacetylase inhibitor anda therapeutically effective amount of a hormone targeted drug. Incertain embodiments, the histone deacetylase inhibitor and the hormonetargeted drug act on the cancer synergistically. In particular cases,the endocrine-regulated cancer has a resistance to the hormone targeteddrug, and the resistance may be de novo resistance or acquiredresistance, for example. In specific embodiments, the histonedeacetylase inhibitor and the hormone targeted drug are administeredsimultaneously or sequentially, for example the histone deacetylaseinhibitor and the hormone targeted drug are administered sequentiallyand the histone deacetylase inhibitor is administered prior to thehormone targeted drug, in specific cases.

In a specific embodiment, the cancer has a resistance to treatment withthe hormone targeted drug when the hormone targeted drug is administeredin an endocrine therapy that does not include the administration of ahistone deacetylase inhibitor. The resistance may be de novo resistanceor acquired resistance, in particular embodiments. In some cases, thecancer responds to treatment with the hormone targeted drug whenadministered in an endocrine therapy that does not include theadministration of a histone deacetylase inhibitor.

In particular embodiments, the endocrine-regulated cancer is breastcancer, prostate cancer, ovarian cancer, or endometrial cancer. Thecancer may be estrogen receptor negative, progesterone receptornegative, androgen receptor positive, or androgen receptor negative, incertain aspects. Furthermore, the hormone targeted drug is an aromataseinhibitor, such as anastrozole, exemestane, letrozole, or combinationsthereof, in certain aspects.

In particular embodiments, prior to treatment with an HDACi andaromatase inhibitor, it is determined that the breast cancer is estrogenreceptor positive. In some embodiments, prior to treatment with an HDACiand aromatase inhibitor, it is determined that the breast cancer isestrogen receptor negative. In some embodiments, prior to treatment withthe HDACi and aromatase inhibitor, the breast cancer is determined to beER−, PR+. In some embodiments, prior to treatment with the HDACi andaromatase inhibitor, the breast cancer is determined to be ER−, PR−, andHer2+. In some embodiments, prior to treatment with the HDACi andaromatase inhibitor, the breast cancer is determined to be ER−, PR− andHer2−.

Also provided herein are methods for assaying a breast cancer cell todetermine if the breast cancer cell is ER+ or ER− and providinginstructions for treating with an HDACi and aromatase inhibitor based onthat determination. Also provided herein are methods for assaying abreast cancer cell to determine if the breast cancer cell is ER−, PR+;ER−, PR−, and Her2+; and/or ER−, PR− and Her2−, and providinginstructions for treating with an HDACi and aromatase inhibitor based onthat determination.

Also provided herein are methods for determining how to treat a breastcancer patient comprising determining if the breast cancer cell is ER+or ER−. Also provided herein are methods for determining how to treat abreast cancer patient comprising determining if the breast cancer cellis ER−, PR+; ER−, PR−, and Her2+; and/or ER−, PR− and Her2−. In somepreferred embodiments, instructions are provided for treatment of apatient with an HDACi and aromatase inhibitor based on the results ofthis determination.

Also provided herein are kits comprising reagents to determine whether acell is estrogen receptor positive and instructions for treatment withan HDACi and aromatase inhibitor. Provided herein are kits comprisingreagents to determine whether a cell is estrogen receptor negative andinstructions for treatment with an HDACi and aromatase inhibitor.Provided herein are kits comprising reagents to determine whether a cellis ER−, PR+ and instructions for treatment with an HDACi and aromataseinhibitor. Provided herein are kits comprising reagents to determinewhether a cell is ER−, PR−, and Her2+ and instructions for treatmentwith an HDACi and aromatase inhibitor. Provided herein are kitscomprising reagents to determine whether a cell is ER−, PR− and Her2−and instructions for treatment with an HDACi and aromatase inhibitor.

In particular embodiments, prior to treatment with an HDACi andanti-androgen, it is determined that the prostate cancer is androgenreceptor negative. In some embodiments, prior to treatment with an HDACiand anti-androgen, it is determined that the prostate cancer is androgenreceptor positive.

Also provided herein are methods for assaying a prostate cancer cell todetermine if the prostate cancer cell is androgen receptor negative orandrogen receptor positive and providing instructions for treating withan HDACi and anti-androgen based on that determination. Also providedherein are methods for determining how to treat a prostate cancerpatient comprising determining if the prostate cancer cell is androgenreceptor negative or androgen receptor positive. In some preferredembodiments, instructions are provided for treatment of a patient withan HDACi and anti-androgen based on the results of this determination.

Also provided herein are kits comprising reagents to determine if a cellis androgen receptor negative and instructions for treatment with anHDACi and anti-androgen. Provided herein are kits comprising reagents todetermine whether a cell is androgen receptor positive and instructionsfor treatment with an HDACi and anti-androgen.

In one embodiment of the invention, there is a method of treating ahormone resistant cancer in a subject having or suspected of having thehormone resistant cancer, comprising the step of administering to thesubject an effective amount of a histone deacetylase inhibitor and anaromatase inhibitor. In some embodiments, the histone deacetylaseinhibitor is PXD-101. In other embodiments, the histone deacetylaseinhibitor is LBH589. In one aspect of the invention, the histonedeacetylase inhibitor is FK228. In another aspect, the histonedeacetylase inhibitor is MGCD-0103. In a specific embodiment, thehistone deacetylase inhibitor is R306465. In one case, the histonedeacetylase inhibitor is PCI-24781. In another case, the histonedeacetylase inhibitor is SB-939. In an additional embodiment of theinvention, the histone deacetylase inhibitor is ITF-2357. In a certainaspect, the histone deacetylase inhibitor is SAHA. In a particularaspect, the histone deacetylase inhibitor is CI-994. In one embodiment,the histone deacetylase inhibitor is MS-275. It is contemplated that anycombination of these histone deacetylase inhibitors may be employed inany method of the invention. In certain embodiments of the invention thearomatase inhibitor may be of any kind, but in a specific embodiment thearomatase inhibitor is anastrozole. In another specific embodiment, thearomatase inhibitor is exemestane. In one aspect of the invention, thearomatase inhibitor is letrozole. In another aspect of the invention,the aromatase inhibitor is formestane. In certain cases, the aromataseinhibitor is testolactone. It is contemplated that any one or morearomatase inhibitors may be employed in any method of the invention. Inparticular embodiments, any combination of one or more of the aromataseinhibitors may be employed with any combination of one or more of thehistone deacetylase inhibitors.

In another embodiment of the invention, there is a method of treating ahormone resistant cancer in a subject having or suspected of having thehormone resistant cancer, comprising the step of administering to thesubject an effective amount of the following: a histone deacetylaseinhibitor; and an aromatase inhibitor. In particular embodiments, thehistone deacetylase inhibitor is PXD-101. In other embodiments, thehistone deacetylase inhibitor is LBH589. In one aspect of the invention,the histone deacetylase inhibitor is FK228. In another aspect, thehistone deacetylase inhibitor is MGCD-0103. In a certain embodiment, thehistone deacetylase inhibitor is R306465. In one case, the histonedeacetylase inhibitor is PCI-24781. In a particular case, the histonedeacetylase inhibitor is SB-939. In an additional embodiment of theinvention, the histone deacetylase inhibitor is ITF-2357. In a certainaspect, the histone deacetylase inhibitor is SAHA. In one aspect, thehistone deacetylase inhibitor is CI-994. In one embodiment, the histonedeacetylase inhibitor is MS-275. It is contemplated that any one or moreof these histone deacetylase inhibitors may be employed in any method ofthe invention. In certain embodiments of the invention the aromataseinhibitor may be of any kind, but in a specific embodiment the aromataseinhibitor is anastrozole. In another specific embodiment, the aromataseinhibitor is exemestane. In certain embodiments of the invention, thearomatase inhibitor is letrozole. In another aspect of the invention,the aromatase inhibitor is formestane. In certain cases, the aromataseinhibitor is testolactone. It is contemplated that any one or morearomatase inhibitors may be employed in any method of the invention. Inparticular embodiments, any combination of one or more of the aromataseinhibitors may be employed with any combination of one or more of thehistone deacetylase inhibitors.

In a certain embodiment of the invention, there is a method of treatinga hormone resistant breast cancer in a subject having or suspected ofhaving the hormone resistant breast cancer, comprising the step ofadministering to the subject an effective amount of the following: ahistone deacetylase inhibitor selected from the group consisting ofSAHA, CI-994, PXD-101, LBH589, FK228, MGCD-0103, R306465, PCI-24781,SB-939, ITF-2357, and MS-275; and an aromatase inhibitor selected fromthe group consisting of anastrozole, exemestane, letrozole, formestane,testolactone, and a combination thereof. In certain embodiments of theinvention, the histone deacetylase inhibitor is PXD-101. In otherembodiments, the histone deacetylase inhibitor is LBH589. In someaspects of the invention, the histone deacetylase inhibitor is FK228. Inanother aspect, the histone deacetylase inhibitor is MGCD-0103. In acertain embodiment, the histone deacetylase inhibitor is R306465. In onecase, the histone deacetylase inhibitor is PCI-24781. In at least onecase, the histone deacetylase inhibitor is SB-939. In an additionalembodiment of the invention, the histone deacetylase inhibitor isITF-2357. In a certain aspect, the histone deacetylase inhibitor isSAHA. In one aspect, the histone deacetylase inhibitor is CI-994. Inspecific embodiments, the histone deacetylase inhibitor is MS-275. It iscontemplated that any one or more of these histone deacetylaseinhibitors may be employed in any method of the invention. In certainembodiments of the invention the aromatase inhibitor may be of any kind,but in a specific embodiment the aromatase inhibitor is anastrozole. Inanother specific embodiment, the aromatase inhibitor is exemestane. Incertain embodiments of the invention, the aromatase inhibitor isletrozole. In another aspect of the invention, the aromatase inhibitoris formestane. In certain cases, the aromatase inhibitor istestolactone. It is contemplated that any one or more aromataseinhibitors may be employed in any method of the invention. In particularembodiments, any combination of one or more of the aromatase inhibitorsmay be employed with any combination of one or more of the histonedeacetylase inhibitors.

In an additional embodiment of the invention, there is a method ofinhibiting growth of a cell comprising contacting the cell with anaromatase inhibitor and a histone deacetylase inhibitor, each in aneffective amount to inhibit growth of the cell, wherein the histonedeacetylase inhibitor is selected from the group consisting of PXD-101,LBH589, FK228, MGCD-0103, R306465, PCI-24781, SB-939, ITF-2357, SAHA andCI-994 and MS-275. In particular embodiments, the histone deacetylaseinhibitor is PXD-101. In other embodiments, the histone deacetylaseinhibitor is LBH589. In one aspect of the invention, the histonedeacetylase inhibitor is FK228. In another aspect, the histonedeacetylase inhibitor is MGCD-0103. In a certain embodiment, the histonedeacetylase inhibitor is R306465. In one case, the histone deacetylaseinhibitor is PCI-24781. In a particular case, the histone deacetylaseinhibitor is SB-939. In an additional embodiment of the invention, thehistone deacetylase inhibitor is ITF-2357. In a certain aspect, thehistone deacetylase inhibitor is SAHA. In one aspect, the histonedeacetylase inhibitor is CI-994. In one embodiment, the histonedeacetylase inhibitor is MS-275. It is contemplated that any one or moreof these histone deacetylase inhibitors may be employed in any method ofthe invention. In certain embodiments of the invention the aromataseinhibitor may be of any kind, but in a specific embodiment the aromataseinhibitor is anastrozole. In another specific embodiment, the aromataseinhibitor is exemestane. In certain embodiments of the invention, thearomatase inhibitor is letrozole. In another aspect of the invention,the aromatase inhibitor is formestane. In certain cases, the aromataseinhibitor is testolactone. It is contemplated that any one or morearomatase inhibitors may be employed in any method of the invention. Inparticular embodiments, any combination of one or more of the aromataseinhibitors may be employed with any combination of one or more of thehistone deacetylase inhibitors.

In an additional embodiment of the invention, there is a method ofinhibiting growth of a cell comprising contacting the cell with anaromatase inhibitor and a histone deacetylase inhibitor, each in aneffective amount to inhibit growth of said cell, wherein the aromataseinhibitor is selected from the group consisting of anastrozole,exemestane, letrozole, formestane, testolactone, and a combinationthereof. In particular embodiments, the histone deacetylase inhibitor isPXD-101. In other embodiments, the histone deacetylase inhibitor isLBH589. In one aspect of the invention, the histone deacetylaseinhibitor is FK228. In another aspect, the histone deacetylase inhibitoris MGCD-0103. In a certain embodiment, the histone deacetylase inhibitoris 8306465. In one case, the histone deacetylase inhibitor is PCI-24781.In a particular case, the histone deacetylase inhibitor is SB-939. In anadditional embodiment of the invention, the histone deacetylaseinhibitor is ITF-2357. In a certain aspect, the histone deacetylaseinhibitor is SAHA. In one aspect, the histone deacetylase inhibitor isCI-994. In one embodiment, the histone deacetylase inhibitor is MS-275.It is contemplated that any one or more of these histone deacetylaseinhibitors may be employed in any method of the invention. In certainembodiments of the invention the aromatase inhibitor may be of any kind,but in a specific embodiment the aromatase inhibitor is anastrozole. Inanother specific embodiment, the aromatase inhibitor is exemestane. Incertain embodiments of the invention, the aromatase inhibitor isletrozole. In another aspect of the invention, the aromatase inhibitoris formestane. In certain cases, the aromatase inhibitor istestolactone. It is contemplated that any one or more aromataseinhibitors may be employed in any method of the invention. In particularembodiments, any combination of one or more of the aromatase inhibitorsmay be employed with any combination of one or more of the histonedeacetylase inhibitors.

In one embodiment of the invention, there is a method of treating ahormone resistant cancer in a subject having or suspected of having thehormone resistant cancer, comprising the step of administering to thesubject an effective amount of the following: a histone deacetylaseinhibitor; and an anti-androgen, wherein the anti-androgen is not a17alpha-hydroxylase-C17,20-lyase inhibitor or a 5-reductase inhibitor.In a specific embodiment, the hormone resistant cancer is prostatecancer. In another specific embodiment, the histone deacetylaseinhibitor is a selective histone deacetylase inhibitor. In a furtherspecific embodiment, the histone deacetylase inhibitor is selected fromthe group consisting of SAHA, CI-994, PXD-101, LBH589, FK228, MGCD-0103,R306465, PCI-24781, SB-939, ITF-2357, and MS-275. In certain aspects,the anti-androgen is selected from the group consisting ofspironolactone (Aldactone, Spiritone; Novo-Spiroton, Spiractin,Verospiron or Berlactone), cyproterone acetate (Androcur, Climen, Diane35, Ginette 35), flutamide (Eulexin), nilutamide (Anandron, Nilandron),bicalutamide (Casodex) and a combination thereof. In some aspects, thehistone deacetylase inhibitor and the anti-androgen are administeredsimultaneously. In other aspects, the histone deacetylase inhibitor isadministered prior to the administration of the anti-androgen. Inparticular cases, the histone deacetylase inhibitor and theanti-androgen act synergistically. In some embodiments, the hormoneresistance is acquired resistance. In particular embodiments, thehistone deacetylase inhibitor is PXD-101. In other embodiments, thehistone deacetylase inhibitor is LBH589. In one aspect of the invention,the histone deacetylase inhibitor is FK228. In another aspect, thehistone deacetylase inhibitor is MGCD-0103. In a certain embodiment, thehistone deacetylase inhibitor is R306465. In one case, the histonedeacetylase inhibitor is PCI-24781. In a particular case, the histonedeacetylase inhibitor is SB-939. In an additional embodiment of theinvention, the histone deacetylase inhibitor is ITF-2357. In a certainaspect, the histone deacetylase inhibitor is SAHA. In one aspect, thehistone deacetylase inhibitor is CI-994. In one embodiment, the histonedeacetylase inhibitor is MS-275. It is contemplated that any one or moreof these histone deacetylase inhibitors may be employed in any method ofthe invention. In certain aspects, the anti-androgen may be of anysuitable kind. In specific embodiments, the anti-androgen isspironolactone (Aldactone, Spiritone; Novo-Spiroton, Spiractin,Verospiron or Berlactone). In certain embodiments, the anti-androgen iscyproterone acetate (Androcur, Climen, Diane 35, Ginette 35). Inparticular embodiments, the anti-androgen is flutamide (Eulexin). Inspecific cases, the anti-androgen is nilutamide (Anandron, Nilandron).In specific cases, the anti-androgen is bicalutamide (Casodex). Inparticular embodiments, any combination of one or more of theanti-androgens may be employed with any combination of one or more ofthe histone deacetylase inhibitors.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings.

FIG. 1A-FIG. 1B show the effect of HDACi (SAHA or MS-275) on the growthof hormone therapy resistant breast cancer cells lines. In particular,FIG. 1A shows combination of HDACi with tamoxifen inhibits growth of ER−breast cancer cells and FIG. 1B shows effect of MS-275 on growth ofletrozole resistant breast cancer cell lines.

FIG. 2 shows the detectable level of aromatase activity in untreatedMDA-MB-231 cells.

FIGS. 3A-3B show the effect of HDAC on aromatase activity. FIG. 3A showsthat exemplary HDAC is stimulate aromatase activity. FIG. 3B shows thatpre-treatment with MS-275 increases aromatase activity of MDA-MB-231cells in a dose-dependent manner.

FIG. 4 demonstrates that HDAC inhibitors upregulate ER and aromatase(CYP19) protein expression.

FIG. 5 shows cell viability following delivery of a combination ofletrozole with HDACi (MS-275 or SAHA).

FIG. 6 demonstrates dose-dependent inhibition by MS-275 of the growth ofbreast cancer xenografts in mice (derived from ER− MDA-MB-231 cells).

FIG. 7 illustrates the effect of MS-275 on tumor and uterine weights ofMDA-MB-231 xenograft-bearing mice.

FIG. 8A shows that MS-275 upregulates tumor ER, AR, and aromatase(CYP19) expression in a dose-dependent manner, and FIG. 8B shows thatMS-275 increases aromatase activity in a dose-dependent manner.

FIGS. 9A-9B demonstrate the effect on tumor volume of singular andcombination therapy for MS-275 and letrozole in MDA-MB-231xenograft-bearing mice.

FIG. 10 is an exemplary cell migration assay in control MDA-MB-231 cellswithout treatment.

FIG. 11 shows the measuring of the width of a parting under a phasecontrast microscope for the cell migration assay.

FIG. 12 is an exemplary measuring of the cell migration assay in thepresence of letrozole.

FIG. 13 is an exemplary measuring of the cell migration assay in thepresence of entinostat (MS-275).

FIG. 14 demonstrates the cell migration assay in the presence of thecombination of entinostat and letrozole.

FIG. 15 graphically illustrates the width of parting in the cellmigration assay and compares the width at 0 hours and 18 hours for thevarious cell treatments.

FIG. 16 graphically illustrates the distance cells migrated across theopen parting during 18 hours with or without various cell treatments inthe cell migration assay.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. For purposes of the presentinvention, the following terms are defined below.

As used herein, the use of the word “a” or “an” when used in conjunctionwith the term “comprising” in the claims and/or the specification maymean “one,” but it is also consistent with the meaning of “one or more,”“at least one,” and “one or more than one.” Some embodiments of theinvention may consist of or consist essentially of one or more elements,method steps, and/or methods of the invention. It is contemplated thatany method or composition described herein can be implemented withrespect to any other method or composition described herein.

The term “acquired resistance” as used herein refers to resistance thatis acquired after at least one treatment with a given agent. Prior tothe at least one treatment, the disorder does not possess a resistanceto the agent (and, as such, the disorder responds to the first treatmentas would a non-resistant disorder). For example, a hormone-resistantcancer is one that initially responds to at least one treatment of ahormone or endrocrine therapy and thereafter develops a resistance tosubsequent treatments of the hormone or endrocrine therapy.

The term “non-androgen responsive” or “androgen resistant” or “androgennegative” refers to a neoplasm that does not utilize an androgen or aderivative thereof or is not sensitive to an androgen or derivativethereof to develop, proliferative and/or metastasize.

The term “de novo resistance” as used herein refers to resistance thatexists prior to treatment with a given agent. Therefore, de novohormone-resistant cancers are resistant to hormone or endocrine therapyprior to the administration of at least one treatment of a hormone orendrocine therapy. In some embodiments, a cancer that is de novoresistant to hormone or endocrine therapy is a cancer that is hormonereceptor negative (e.g., estrogen receptor negative or progesteronereceptor negative).

The term “effective amount” or “therapeutically effective amount” asused herein is defined as an amount of the agent that will decrease,reduce, inhibit or otherwise abrogate the growth of a neoplasm, induceapoptosis, inhibit angiogenesis of a neoplasm, inhibit metastasis, orinduce cytotoxicity in a neoplasm. Thus, an effective amount is anamount sufficient to detectably and repeatedly ameliorate, reduce,minimize or limit the extent of the disease or its symptoms.

The term “disease-free survival” as used herein is defined as a timebetween the first diagnosis and/or first surgery to treat a cancerpatient and a first reoccurrence. For example, a disease-free survivalis “low” if the cancer patient has a first reoccurrence within fiveyears after tumor resection, and more specifically, if the cancerpatient has less than about 55% disease-free survival over 5 years. Forexample, a high disease-free survival refers to at least about 55%disease-free survival over 5 years.

The term “endocrine-regulated cancer” as used herein refers to cancersthat progress, at least at some stage of their progression, in a mannerdependent on the expression of a hormone or a hormone receptor,including, by way of non-limiting example, estrogen, progesterone,and/or the receptors thereof.

The term “HDAC inhibitor” as used herein refers to a compound that hasthe ability to inhibit histone deacetylase activity. This therapeuticclass is able to block angiogenesis and cell cycling, and promoteapoptosis and differentiation. HDAC inhibitors both display targetedanticancer activity by themselves and improve the efficacy of existingagents as well as other new targeted therapies.

The term “hormone-resistant cancer” as used herein refers to a cancerthat has a decreased or eliminated response to a hormone therapy orendocrine therapy when compared to a non-hormone-resistant cancer. Froma biological and clinical standpoint, several patterns of resistance canbe distinguished: A) tumors that are inherently insensitive to endocrinereceptor (e.g., estrogen receptor) targeting despite endocrine receptorexpression (pan-endocrine therapy resistance or de novo resistance); B)tumors that are hormone dependent but resistant to one or more specificendocrine therapies (agent-selective resistance; for example respondedto tamoxifen but not aromatase inhibitor); and C) tumors that initiallyrespond to endocrine therapy but subsequently progress (acquiredresistance). All types of resistance are included herein. In someembodiments, the hormone-resistant cancer is a cancer that ishormone-resistant prior to the administration of a hormone or endrocinetherapy (i.e., it is de novo hormone-resistant). In other embodiments,the hormone-resistant cancer is a cancer that is initially nothormone-resistant, but becomes hormone-resistant after at least onetreatment of a hormone or endocrine therapy.

The term “hormone therapy” or “endocrine therapy” as used herein isdefined as a treatment pertaining to blocking or removing hormones. Thetreatment may remove the gland that synthesizes the hormone or theprohormone, block or inhibit hormone synthesis, or prevent or inhibitthe hormone from binding to its receptor, or down-regulate or degradethe hormone receptor.

The term “hormone agent” or “hormone targeted drug” is an agent thatblocks or inhibits hormone synthesis, prevents or inhibits the hormonefrom binding to its receptor, or down-regulates or degrades the hormonereceptor.

The term “endocrine therapy-resistant” or “hormone resistant” as usedherein is defined as a subject receiving an endocrine therapy orhormonal therapy and lacks demonstration of a desired physiologicaleffect, such as a therapeutic benefit, from the administration of thetherapy.

The term “estrogen-receptor positive” as used herein refers to cancersthat do have estrogen receptors while those breast cancers that do notpossess estrogen receptors are “estrogen receptor-negative.”

The term “neoplasm” as used herein refers to an abnormal formation oftissue, for example, a tumor. One of skill in the art realizes that aneoplasm encompasses benign tumors and/or malignant tumors. Yet further,as used herein the terms “neoplasm” and “tumor” are interchangeable.

As used herein the term “refractory” means a cancer that does notrespond to treatment. The cancer may be resistant at the beginning oftreatment or it may become resistant during treatment.

The term “subject” as used herein, is taken to mean any mammaliansubject to which a composition of the present invention is administeredaccording to the methods described herein. In a specific embodiment, themethods of the present invention are employed to treat a human subject.Another embodiment includes treating a human subject suffering from abreast neoplasm.

The terms “synergistic” or “synergistically” as used herein refer to twoor more compounds providing a therapeutic effect that is greater thanthe sum of the therapeutic effects of the two compounds provided astherapy alone.

The term “therapeutically effective amount” as used herein refers to anamount that results in an improvement or remediation of the symptoms ofthe disease or condition.

The term “therapeutic benefit” as used herein refers to anything thatpromotes or enhances to a significant extent the well-being of thesubject with respect to the medical treatment of his condition, whichincludes treatment of pre-cancer, cancer, and hyperproliferativediseases. A list of nonexhaustive examples of this includes extension ofthe subject's life by any period of time, decrease or delay in theneoplastic development of the disease, decrease in hyperproliferation,reduction in tumor growth, delay of metastases, reduction in cancer cellor tumor cell proliferation rate, and a decrease in pain to the subjectthat can be attributed to the subject's condition. In a specificembodiment, a therapeutic benefit refers to reversing de novo hormonetherapy-resistance or preventing the patient from acquiring an hormonetherapy-resistance.

II. Methods of Treatment

In a particular aspect, the present invention provides methods for thetreatment of hormone resistant cancers. The present invention relates toa histone deacetylase inhibitor (HDACi) and targeted hormone agents ordrugs that, in some embodiments, shows unexpected, potent synergisticanti-cancer activity. In certain embodiments, the HDACi increases thesensitivity of the cells to the targeted hormone agent, thus, thiscombination of HDACi and traditional hormone therapy can be used totreat cancers that are typically not treatable with hormone therapy.Thus, the present invention provides a treatment for hormone resistantcancers in a subject, in particular cases.

A. Cancer Types

In certain embodiments, the HDACi and a targeted hormone agent or drugare administered to a cell. Cells that are encompassed by the presentinvention include, for example, epithelial cancer cells. In someembodiments, the cancer cell is a hormone resistant cancer cell.

1. Breast Cancer

Cells that are encompassed by the present invention include, but are notlimited to, breast cells. More specifically, the breast cell is a cancercell, a non-cancerous cell or a benign hyperplastic cell. A breastcancer cell may include cells that are drug-resistant, primary cancercells and/or metastatic cancer cells, for example.

In certain aspects, an effective amount of a HDACi and a targetedhormone agent, e.g., an aromatase inhibitor, may be administered to asubject suffering from breast cancer, more specifically, estrogenreceptor negative breast cancer (ER−) or hormone resistant breastcancer. In certain embodiments the HDACi increases aromatase expressionand/or activity, thus making the tumors or neoplams more sensitive toaromatase inhibitors in addition to antiestrogens. The effectiveness ofthe therapy according to the present invention can be determined in thetreatment of estrogen cancer by diagnostic methods that are known andused in the art, for example, but not limited to, a mammogram, anultrasound, a biopsy, etc.

Other embodiments include methods for inhibiting development of breastcancer in a subject at risk, inhibiting breast cancer metastasis in asubject with primary breast cancer, and/or inhibiting breast cancerprogression. Also within the scope of the invention is a method oftreating benign breast hyperplasia in a human subject afflicted withbenign breast hyperplasia comprising administering a HDACi and atargeted hormone agent thereof to the subject in an amount and durationsufficient to result in cell killing or decreases in cell viability.

Still further, other embodiments can include a method of increasing thesensitivity of estrogen negative cancer cells by administering an HDACi.By administering the HDACi to these estrogen negative cancer cells, theHDACi increases expression of estrogen receptors thereby increasing thesensitivity of these cells to hormone therapy. Thus, the combination ofHDACi and endocrine therapies or hormonal therapies, e.g., aromataseinhibitors, can be used to inhibit or reduce estrogen negative typecancer cells thereby treating a hormone therapy resistant cancer withstandard hormone therapies in combination with HDAC inhibitors.

Another embodiment of the present invention can comprise a method ofinhibiting or decreasing cancer cell growth by administering to the cella HDACi in combination with an aromatase inhibitor. The HDACi increasesaromatase expression and/or activity, thus making the tumors or neoplamsmore sensitive to aromatase inhibitors in addition to antiestrogens,thus inhibiting or decreasing cell growth or proliferation.

Non-limiting examples of endocrine therapies that are contemplated bythe present invention include tamoxifen, raloxifene, toremifene or otherSERMs (selective estrogen-receptor modulators). Tamoxifen has been themost commonly prescribed drug to treat breast cancer since its approvalby the U.S. Food and Drug Administration (FDA) in the 1970s. Tamoxifenis an anti-estrogen and works by competing with the hormone estrogen tobind to estrogen receptors in breast cancer cells. Tamoxifen has beenshown to reduce the risk of recurrence of an original cancer and therisk of developing new cancers by working against the effects ofestrogen on breast cancer cells. A pharmaceutical composition comprisingtamoxifen is generally administered as an oral composition such as apill or capsule. Tamoxifen belongs to a class of agents known asselective estrogen receptor modulators. These agents display estrogenantagonist activity on some genes and agonist activity on others.

In other specific embodiments, the endocrine therapy comprises goserelinacetate, leuprolide acetate, exemestane, megestrol, toremifene,fulvestrant, a nonsteroidal or a steroidal aromatase inhibitorincluding, for example, anastrozole, exemestane and letrozole.Fulvestrant has demonstrated an ability to destroy estrogen receptors inbreast cancer cells, for example.

2. Prostate Cancer

In certain aspects, an effective amount of a HDACi and a targetedhormone agent, e.g., as anti-androgen, may be administered to a subjectsuffering from prostate cancer, more specifically, recurrent prostatecancer, more specifically, hormone resistant prostate cancer. Theeffectiveness of the therapy according to the present invention can bedetermined in the treatment of prostate cancer by diagnostic methodsthat are known and used in the art, for example, but not limited to,analysis of prostate specific antigen (PSA), a prostate biopsy, a rectalexam, or analysis of PSA and rectal exam.

Other embodiments include methods for inhibiting development of prostatecancer in a subject at risk, inhibiting prostate cancer metastasis in asubject with primary prostate cancer, and/or inhibiting prostate cancerprogression in subjects.

Also within the scope of the invention is a method of treating benignprostate hyperplasia in a human subject afflicted with benign prostatehyperplasia comprising administering a HDACi and a targeted hormoneagent thereof to the subject in an amount and duration sufficient toresult in cell killing or decreases in cell viability. The levels ofprostate specific antigen (PSA) produced by the hyperplastic cells couldalso be stabilized or reduced upon treatment with a HDACi and a targetedhormone agent.

Still further, other embodiments can include a method of increasing thesensitivity of androgen negative cancer cells by administering an HDACi.By administering the HDACi to these androgen negative cancer cells, theHDACi increases expression of androgen receptors thereby increasing thesensitivity of these cells to hormone therapy. Thus, HDACi andanti-androgens in combination to inhibit or reduce androgen negativetype cancer cells thereby treating a hormone therapy resistant cancerwith standard hormone therapies in combination with HDAC inhibitors.

Still other embodiments can include a method of increasing thesensitivity of androgen-receptor positive (AR+) cancer cells byadministering an HDACi. By administering the HDACi to these AR+ cancercells, the HDACi down regulates expression of the androgen receptorsthereby increasing the sensitivity of these cells to hormone therapy. Insome embodiments, the cancer cells are AR+ and the HDACi resensitizesthe cells to an anti-androgen.

In further embodiments, anti-androgens are used in combination withHDACi to treat ER− tumors. In some embodiments, the treatment with theanti-androgen renders the cells ER+.

In other embodiments, anti-estrogens, such as tamoxifen, are used incombination with an HDACi described herein to treat lung cancer.

B. HDACi

Inhibitors of histone deacetylase inhibitors induce hyperacetylation ofhistones that modulate chromatin structure and gene expression. Theseinhibitors also induce growth arrest, cell differentiation, andapoptosis of tumor cells. Recently it was reported that HDACi canrestore the expression of functional ERα to ER− breast cancer cells(Ferguson et al. 2004; Sharma et al. 2006; Yang et al. 2000; Keen et al.2003). The discovery of recruitment of histone deacetylase (HDAC)enzymes in cancer has provided a rationale for using inhibition of HDACactivity to release transcriptional repression as viable option towardachieving eventual therapeutic benefit (Vigushin et al. 2002). Histonedeacetylase inhibitors (HDACis) block deacetylation function, causingcell cycle arrest, differentiation, and/or apoptosis of many tumors(Vigushin et al. 2002). Silencing of genes that affect growth anddifferentiation has been shown to occur by aberrant DNA methylation inpromoter region and by changes in chromatin structure that involvehistone deacetylation. Recent studies have established a link betweenoncogene-mediated suppression of transcription and recruitment of HDACinto nuclear complex. HDACi such as butyric acid (BA), 4-phenylbutyricacid and trichostatin A reverse this suppression by specific inhibitionof HDAC activity, leading to histone hyperacetylation, chromatinrelaxation, and enhanced transcription.

The HDACs are a family including at least eighteen enzymes, grouped inthree classes (Class I, II and III). Class I HDACs include, but are notlimited to, HDACs 1, 2, 3, 8 and 11. Class I HDACs can be found in thenucleus and are believed to be involved with transcriptional controlrepressors. Class II HDACs include, but are not limited to, HDACs 4, 5,6, 7, and 9 and can be found in both the cytoplasm as well as thenucleus. Class III HDACs are believed to be NAD dependent proteins andinclude, but are not limited to, members of the Sirtuin family ofproteins. Non-limiting examples of sirtuin proteins include SIRT1-7. Asused herein, the term “selective HDAC” refers to an HDAC inhibitor thatdoes not substantially interact with all three HDAC classes. The term“Class I Selective HDAC” refers to an HDAC inhibitor that does notsubstantially interact with Class II or Class III HDACs.

In various embodiments, the HDAC inhibitor is a non-selective HDACinhibitor. In specific embodiments, the non-selective HDAC inhibitor is,by way of non-limiting example, N′-hydroxy-N-phenyl-octanediamide(suberoylanilide hydroxamic acid, SAHA), pyroxamide, CBHA, trichostatinA (TSA), trichostatin C, salicylihydroxamic acid (SBHA), azelaicbihydroxamic acid (ABHA), azelaic-1-hydroxamate-9-analide (AAHA),depsipeptide, FK228, 6-(3-chlorophenylureido) carpoic hydroxamic acid(3Cl-UCHA), oxamflatin, A-161906, scriptaid, PXD-101 (Belinostat orN-hydroxy-3-[3-](phylamino)sulfonyl phenyl]-2-propenamide), LAQ-824,CHAP, MW2796, LBH589 (Panobinostat), CI-994(“4-acetylamino-N-(2′-aminophenyl)-benzamide”), R306465, ITF2357,PCI-24781, SB-939, or MW2996.

In certain embodiments, the HDAC inhibitor inhibits at least one ofHDAC-1, HDAC-2, HDAC-3, HDAC-8, or HDAC-11. In a specific embodiment,the HDAC inhibitor inhibits HDAC-1. In another embodiment, the HDACinhibitor inhibits HDAC-2. In yet another embodiment, the HDAC inhibitorinhibits HDAC-3. In another embodiment, the HDAC inhibitor inhibitsHDAC-8. In still another embodiment, the HDAC inhibitor inhibitsHDAC-11. In other embodiments, the HDAC inhibitor inhibits HDAC-1,HDAC-2, HDAC-3 and HDAC-11.

In specific embodiments of the present invention, the Class I selectiveHDAC inhibitor is, by way of non-limiting example, MGCD-0103(N-(2-amino-phenyl)-4-[(4-pyridin-3-yl-pyrimidin-2-ylamino)-methyl]-benzamide),MS-275 (N-(2-aminophenyl)-4-(N-(pyridin-3-ylmethoxycarbonyl)aminomethyl)benzamide, SNDX-275), spiruchostatin A, SK7041, SK7068 and 6-aminonicotinamides.

In various embodiments of the present invention, the HDAC inhibitors areused to increase the sensitivity or to sensitize hormone resistantcancer cells to hormonal therapy. In some embodiments, the HDACi isselected from one of the following groups: The short-chain fatty acids(e.g., butyrate and phenylbutyrate, isovalerate, valproate, 4-phenylbutyrate (4-PBA), phenylbutyrate propionate, butyaramide,isobutyaramide, phenylacetate, 3-bromopropionate, tributyrin, valproicacid, and Pivanex); hydroxamic acids (e.g., the trichostatins such asTSA and TSC, suberoylanilide hydroxamic acid (“SAHA”) and itsderivatives, Oxamflatin, azelaic biyhydroxamic acid (“ABHA”),azelaic-1-hydroxamate-9-anilide (“AAHA”), suberoyl bishydroxamic acid(“SBHA”), m-carboxycinnamic acid bishydroxamide (“CBHA”), pyrozamide,salicylbishyudoxamic acid, Scriptaid, Pyroxamide, Propenamides, LBH589,CHAP, MY29996, MW2976, and any of the hydroximic acids disclosed in U.S.Pat. Nos. 5,369,108; 5,932,616; 5,5,700,811; 6,087,367; and 6,511,990);epoxyketone-containing cyclic tetrapeptides (e.g., trapoxins,depeudecin, depsipeptide FK228, FR 225497, Apicidin, cyclictetrapeptide, Apicidin Ia, Apicidin Ib, Apicidin Ic, Apicidin IIa,Apicidin III), a cycli tetrapeptide containing a2-amino-8-oxo-9,10-epoxy-decanoyl moitey, a cyclic peptide without the2-amino-8-oxo-9, 10 epoxy-decanoyl moity, HC-toxin, Chlamydocin,Diheteropeptin, WF-3161, Cyl-1 and Cyl-2); non-epoxyketone-containingcyclic tetrapeptides (e.g, FR901228, Apicidin,cyclic-hydroxamic-acid-containing peptides (CHAPs); benzamides (e.g.,MS-275 (MS-27-275, SNDX-275), N-acetyldinaline, CI-994, MGCD0103, otherbenzamide analogs); and other miscellaneous structures (e.g., Savicol,Bacecca, MG98, Depudecin, Organosulfur compounds).

C. Treatment Regimen

Treatment methods will involve treating an individual with an effectiveamount of a HDACi and a targeted hormone agent or drug. An effectiveamount is described, generally, as that amount sufficient to detectablyand repeatedly to ameliorate, reduce, minimize or limit the extent of adisease or, its symptoms. More specifically, it is envisioned that thetreatment with the HDACi and targeted hormone agent will kill cells,inhibit cell growth, inhibit metastasis, decrease tumor size andotherwise reverse or reduce the malignant phenotype of tumor cells.

An effective amount of HDACi and a targeted hormone agent or drug thatmay be administered to a cell includes a dose of about 0.1 μM to about100 μM. More specifically, doses of HDACi and a target hormone agent ordrug to be administered are from about 0.1 μM to about 1 μM; about 1 μMto about 5 μM; about 5 μM to about 10 μM; about 10 μM to about 15 μM;about 15 μM to about 20 μM; about 20 μM to about 30 μM; about 30 toabout 40 μM; about 40 μM to about 50 μM; about 50 μM to about 60 μM;about 60 μM to about 70 μM; about 70 μM to about 80 μM; about 80 μM toabout 90 μM; and about 90 μM to about 100 μM. Of course, all of theseamounts are exemplary, and any amount in-between these points is alsoexpected to be of use in the invention.

The effective amount or “therapeutically effective amounts” of the HDACiand a targeted hormone agent or drug to be used are those amountseffective to produce beneficial results, particularly with respect tocancer treatment, in the recipient animal or patient. Such amounts maybe initially determined by reviewing the published literature, byconducting in vitro tests or by conducting metabolic studies in healthyexperimental animals. Before use in a clinical setting, it may bebeneficial to conduct confirmatory studies in an animal model,preferably a widely accepted animal model of the particular disease tobe treated. Preferred animal models for use in certain embodiments arerodent models, which are preferred because they are economical to useand, particularly, because the results gained are widely accepted aspredictive of clinical value.

As is well known in the art, a specific dose level of active compoundssuch as HDACi and a targeted hormone agent or drug any particularpatient depends upon a variety of factors including the activity of thespecific compound employed, the age, body weight, general health, sex,diet, time of administration, route of administration, rate ofexcretion, drug combination, and the severity of the particular diseaseundergoing therapy. The person responsible for administration willdetermine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

A therapeutically effective amount of HDACi and a targeted hormone agentor drug as a treatment varies depending upon the host treated and theparticular mode of administration. However, it is noted that the dosagesdescribed in the Examples provided herein provide guidance to theskilled artisan for use in a mammal. For example, in some embodiments itis recognized in the art that dosage by weight in a mouse is usuallygreater than dosage by weight in a human due to differences inmetabolism of at least certain drugs. Therefore, in some cases thedosages described in the Examples may be used as guidance for themaximum dosage that may be utilized for a human, and in certain aspectsa certain fold difference less than these dosages is employed in ahuman. In a specific example, the dosage is an order of magnitude lessfor a human than a mouse. In certain embodiments, when a combination ofHDACi and aromatase inhibitor are employed, the dosages may be the sameor less or more than when delivered alone.

With the knowledge of one of skill in the art and the teaching providedherein, dosages for aromatase inhibitors useful in the present inventioncan be determined. Specific non-limiting examples of doses of aromataseinhibitors include the following: 1 mg of Arimidex administered once aday, 2.5 mg of Femara administered once a day, 25 mg of Teslacadministered once a day, and 250 mg Cytadren administered once a day.

Also with the knowledge of one of skill in the art and the teachingprovided herein, dosages for anti-androgens useful in the presentinvention can be determined. Specific non-limiting examples of doses ofanti-androgens useful in the present invention include: 25, 50 or 100mgs Aldactone administered once daily, 300 mgs Androcurus administeredonce daily, 125 mgs Eulexin administered three times a day, and 150 mgsNilandron administered twice daily or once daily.

In one embodiment of the invention the dose range of the HDACi and atargeted hormone agent or drug will be about 0.5 mg/kg body weight toabout 500 mg/kg body weight. The term “body weight” is applicable whenan animal is being treated. When isolated cells are being treated, “bodyweight” as used herein should read to mean “total cell weight”. The term“total weight” may be used to apply to both isolated cell and animaltreatment. All concentrations and treatment levels are expressed as“body weight” or simply “kg” in this application are also considered tocover the analogous “total cell number” and “total weight”concentrations. However, those of skill will recognize the utility of avariety of dosage range, for example, 1 mg/kg body weight to 450 mg/kgbody weight, 2 mg/kg body weight to 400 mg/kg body weight, 3 mg/kg bodyweight to 350 mg/kg body weight, 4 mg/kg body weight to 300 mg/kg bodyweight, 5 mg/kg body weight to 250 mg/kg body weight, 6 mg/kg bodyweight to 200 mg/kg body weight, 7 mg/kg body weight to 150 mg/kg bodyweight, 8 mg/kg body weight to 100 mg/kg body weight, or 9 mg/kg bodyweight to 50 mg/kg body weight. Further, those of skill will recognizethat a variety of different dosage levels will be of use, for example, 1mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 12.5mg/kg, 15 mg/kg, 17.5 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100mg/kg, 120 mg/kg, 140 mg/kg, 150 mg/kg, 160 mg/kg, 180 mg/kg, 200 mg/kg,225 mg/kg, 250 mg/kg, 275 mg/kg, 300 mg/kg, 325 mg/kg, 350 mg/kg, 375mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 700 mg/kg,750 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, 1250 mg/kg, 1500 mg/kg,1750 mg/kg, 2000 mg/kg, 2500 mg/kg, and/or 3000 mg/kg. Of course, all ofthese dosages are exemplary, and any dosage in-between these points isalso expected to be of use in the invention. Any of the above dosageranges or dosage levels may be employed for HDACi and targeted hormoneagent or drug.

Administration of a HDACi and a targeted hormone agent to a patient orsubject will follow general protocols for the administration ofchemotherapeutics, taking into account the toxicity, if any. It isexpected that the treatment cycles would be repeated as necessary. Italso is contemplated that various standard therapies, as well assurgical intervention, may be applied in combination with the describedtherapy.

The treatments may include various “unit doses.” Unit dose is defined ascontaining a predetermined quantity of the HDACi and targeted hormoneagent calculated to produce the desired responses in association withits administration, i.e., the appropriate route and treatment regimen.The quantity to be administered, and the particular route andformulation, are within the skill of those in the clinical arts. Also ofimport is the subject to be treated, in particular, the state of thesubject and the protection desired. A unit dose need not be administeredas a single injection but may comprise continuous infusion, for examplebroken into a set of injections, over a set period of time.

According to the present invention, one may treat the cancer by directlyinjection a tumor with the HDACi and targeted hormone agent.Alternatively, the tumor may be infused or perfused with the compositionusing any suitable delivery vehicle. Local or regional administration,with respect to the tumor, also is contemplated. More preferably,systemic administration or oral administration may be performed.Continuous administration also may be applied where appropriate, forexample, where a tumor is excised and the tumor bed is treated toeliminate residual, microscopic disease. Delivery via syringe orcatherization is preferred. Such continuous perfusion may take place fora period from about 1-2 hours, to about 2-6 hours, to about 6-12 hours,to about 12-24 hours, to about 1-2 days, to about 1-2 wk or longerfollowing the initiation of treatment. Generally, the dose of thetherapeutic composition via continuous perfusion will be equivalent tothat given by a single or multiple injections, adjusted over a period oftime during which the perfusion occurs. For tumors of >4 cm, the volumeto be administered will be about 4-10 ml (preferably 10 ml), while fortumors of <4 cm, a volume of about 1-3 ml will be used (preferably 3ml). Multiple injections delivered as single dose comprise about 0.1 toabout 0.5 ml volumes.

In certain embodiments, the tumor being treated may not, at leastinitially, be resectable. Treatments with a HDACi and a targeted hormoneagent may increase the resectability of the tumor due to shrinkage atthe margins or by elimination of certain particularly invasive portions.Following treatments, resection may be possible. Additional treatmentssubsequent to resection will serve to eliminate microscopic residualdisease at the tumor site.

III. Combined Cancer Therapy with Other Anticancer Agents

In the context of the present invention, it is contemplated that HDACiand the hormonal therapy thereof may be used in combination with anadditional therapeutic agent to more effectively treat the cancer.Anticancer agents may include but are not limited to, radiotherapy,chemotherapy, gene therapy, or immunotherapy that targets cancer/tumorcells.

When an additional therapeutic agent is administered, as long as thedose of the additional therapeutic agent does not exceed previouslyquoted toxicity levels, the effective amounts of the additionaltherapeutic agent may simply be defined as that amount effective toinhibit and/or reduce the cancer growth when administered to an animalin combination with the HDACi and the hormonal therapy agents. This maybe easily determined by monitoring the animal or patient and measuringthose physical and biochemical parameters of health and disease that areindicative of the success of a given treatment. Such methods are routinein animal testing and clinical practice.

To kill cells, induce cell-cycle arrest, inhibit cell growth, inhibitmetastasis, inhibit angiogenesis or otherwise reverse or reduce themalignant phenotype of cancer cells, using the methods and compositionsof the present invention, one would generally contact a cell with HDACiand the hormonal therapy agent thereof in combination with an additionaltherapeutic agent. These compositions would be provided in a combinedamount effective to inhibit cell growth and/or induce apoptosis in thecell. This process may involve contacting the cells with HDACi and thehormonal therapy agent thereof in combination with an additionaltherapeutic agent or factor(s) at the same time. This may be achieved bycontacting the cell with a single composition or pharmacologicalformulation that includes both agents, or by contacting the cell withtwo distinct compositions or formulations, at the same time, wherein onecomposition includes the HDACi and the hormonal therapy agent thereofand the other includes the additional agent.

Alternatively, treatment with HDACi and the hormonal therapy agent mayprecede or follow the additional agent treatment by intervals rangingfrom minutes to weeks. In embodiments where the additional agent isapplied separately to the cell, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the agent would still be able to exert anadvantageously combined effect on the cell. In such instances, it iscontemplated that one would contact the cell with both modalities withinabout 12-24 hr of each other and, more preferably, within about 6-12 hrof each other, with a delay time of only about 12 hr being mostpreferred. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several days (2, 3,4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse betweenthe respective administrations.

It also is conceivable that more than one administration of either TGHDACi and the hormonal therapy agent in combination with an additionaltherapeutic agent such as anticancer agent will be desired. Variouscombinations may be employed, where HDACi and the hormonal therapy agentthereof is “A” and the additional therapeutic agent is “B”, asexemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/B

A. Chemotherapeutic Agents

In some embodiments of the present invention chemotherapy may beadministered, as is typical, in regular cycles. A cycle may involve onedose, after which several days or weeks without treatment ensues fornormal tissues to recover from the drug's side effects. Doses may begiven several days in a row, or every other day for several days,followed by a period of rest. If more than one drug is used, thetreatment plan will specify how often and exactly when each drug shouldbe given. The number of cycles a person receives may be determinedbefore treatment starts (based on the type and stage of cancer) or maybe flexible, in order to take into account how quickly the tumor isshrinking. Certain serious side effects may also require doctors toadjust chemotherapy plans to allow the patient time to recover.

Chemotherapeutic agents that may be used in combination with the presentinvention, include, but are not limited to cisplatin (CDDP),carboplatin, procarbazine, mechlorethamine, cyclophosphamide,camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea,dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin,mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptorbinding agents, gemcitabien, navelbine, farnesyl-protein tansferaseinhibitors, transplatinum, 5-fluorouracil and methotrexate, or anyanalog or derivative variant of the foregoing.

B. Radiotherapeutic Agents

Radiotherapeutic agents may also be use in combination with thecompounds of the present invention in treating a cancer. Such factorsthat cause DNA damage and have been used extensively include what arecommonly known as γ-rays, X-rays, and/or the directed delivery ofradioisotopes to tumor cells. Other forms of DNA damaging factors arealso contemplated such as microwaves and UV-irradiation. It is mostlikely that all of these factors effect a broad range of damage on DNA,on the precursors of DNA, on the replication and repair of DNA, and onthe assembly and maintenance of chromosomes. Dosage ranges for X-raysrange from daily doses of 50 to 200 roentgens for prolonged periods oftime (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosageranges for radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe neoplastic cells.

C. Immunotherapeutic Agents

Immunotherapeutics may also be employed in the present invention incombination with HDACi and the hormonal therapy agent in treatingcancer. Immunotherapeutics, generally, rely on the use of immuneeffector cells and molecules to target and destroy cancer cells. Theimmune effector may be, for example, an antibody specific for somemarker on the surface of a tumor cell. The antibody alone may serve asan effector of therapy or it may recruit other cells to actually effectcell killing. The antibody also may be conjugated to a drug or toxin(chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussistoxin, etc.) and serve merely as a targeting agent. Alternatively, theeffector may be a lymphocyte carrying a surface molecule that interacts,either directly or indirectly, with a tumor cell target. Variouseffector cells include cytotoxic T cells and NK cells.

Generally, the tumor cell must bear some marker that is amenable totargeting, i.e., is not present on the majority of other cells. Manytumor markers exist and any of these may be suitable for targeting inthe context of the present invention. Common tumor markers includecarcinoembryonic antigen, prostate specific antigen, urinary tumorassociated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG,Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B and p155.

D. Inhibitors of Cellular Proliferation

The tumor suppressor oncogenes function to inhibit excessive cellularproliferation. The inactivation of these genes destroys their inhibitoryactivity, resulting in unregulated proliferation. The tumor suppressorsp53, p16 and C-CAM are described below.

High levels of mutant p53 have been found in many cells transformed bychemical carcinogenesis, ultraviolet radiation, and several viruses. Thep53 gene is a frequent target of mutational inactivation in a widevariety of human tumors and is already documented to be the mostfrequently mutated gene in common human cancers. It is mutated in over50% of human NSCLC (Hollstein et al., 1991) and in a wide spectrum ofother tumors.

The p53 gene encodes a 393-amino acid phosphoprotein that can formcomplexes with host proteins such as large-T antigen and E1B. Theprotein is found in normal tissues and cells, but at concentrationswhich are minute by comparison with transformed cells or tumor tissue.

Wild-type p53 is recognized as an important growth regulator in manycell types. Missense mutations are common for the p53 gene and areessential for the transforming ability of the oncogene. A single geneticchange prompted by point mutations can create carcinogenic p53. Unlikeother oncogenes, however, p53 point mutations are known to occur in atleast 30 distinct codons, often creating dominant alleles that produceshifts in cell phenotype without a reduction to homozygosity.Additionally, many of these dominant negative alleles appear to betolerated in the organism and passed on in the germ line. Various mutantalleles appear to range from minimally dysfunctional to stronglypenetrant, dominant negative alleles (Weinberg, 1991).

Another inhibitor of cellular proliferation is p16. The majortransitions of the eukaryotic cell cycle are triggered bycyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4(CDK4), regulates progression through the G1. The activity of thisenzyme may be to phosphorylate Rb at late G1. The activity of CDK4 iscontrolled by an activating subunit, D-type cyclin, and by an inhibitorysubunit, the p16INK4 has been biochemically characterized as a proteinthat specifically binds to and inhibits CDK4, and thus may regulate Rbphosphorylation (Serrano et al., 1993; Serrano et al., 1995). Since thep16INK4 protein is a CDK4 inhibitor (Serrano, 1993), deletion of thisgene may increase the activity of CDK4, resulting inhyperphosphorylation of the Rb protein. p16 also is known to regulatethe function of CDK6.

p16INK4 belongs to a newly described class of CDK-inhibitory proteinsthat also includes p16B, p19, p21WAF1, and p27KIP1. The p16INK4 genemaps to 9p21, a chromosome region frequently deleted in many tumortypes. Homozygous deletions and mutations of the p16INK4 gene arefrequent in human tumor cell lines. This evidence suggests that thep16INK4 gene is a tumor suppressor gene. This interpretation has beenchallenged, however, by the observation that the frequency of thep16INK4 gene alterations is much lower in primary uncultured tumors thanin cultured cell lines (Caldas et al., 1994; Cheng et al., 1994;Hussussian et al., 1994; Kamb et al., 1994; Kamb et al., 1994; Mori etal., 1994; Okamoto et al., 1994; Nobori et al., 1995; Orlow et al.,1994; Arap et al., 1995). Restoration of wild-type p16INK4 function bytransfection with a plasmid expression vector reduced colony formationby some human cancer cell lines (Okamoto, 1994; Arap, 1995).

Other genes that may be employed according to the present inventioninclude Rb, mda-7, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73,VHL, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16 fusions, p21/p27fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras,myc, neu, raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, genesinvolved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF,or their receptors) and MCC.

E. Regulators of Programmed Cell Death

Apoptosis, or programmed cell death, is an essential process in cancertherapy (Ken et al., 1972). The Bcl-2 family of proteins and ICE-likeproteases have been demonstrated to be important regulators andeffectors of apoptosis in other systems. The Bcl-2 protein, discoveredin association with follicular lymphoma, plays a prominent role incontrolling apoptosis and enhancing cell survival in response to diverseapoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, 1985; Clearyet al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). Theevolutionarily conserved Bcl-2 protein now is recognized to be a memberof a family of related proteins, which can be categorized as deathagonists or death antagonists.

Members of the Bcl-2 that function to promote cell death such as, Bax,Bak, Bik, Bim, Bid, Bad and Harakiri, are contemplated for use incombination with HDACi and a homonal therapy agent thereof in treatingcancer.

F. Surgery

It is further contemplated that a surgical procedure may be employed inthe present invention. Approximately 60% of persons with cancer willundergo surgery of some type, which includes preventative, diagnostic orstaging, curative and palliative surgery. Curative surgery includesresection in which all or part of cancerous tissue is physicallyremoved, excised, and/or destroyed. Tumor resection refers to physicalremoval of at least part of a tumor. In addition to tumor resection,treatment by surgery includes laser surgery, cryosurgery,electrosurgery, and miscopically controlled surgery (Mohs' surgery). Itis further contemplated that the present invention may be used inconjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

G. Other Agents

It is contemplated that other agents may be used in combination with thepresent invention to improve the therapeutic efficacy of treatment.These additional agents include immunomodulatory agents, agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion, oragents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers. Immunomodulatory agents include tumor necrosisfactor; interferon alpha, beta, and gamma; IL-2 and other cytokines;F42K and other cytokine analogs; or MIP-1, MIP-1beta, MCP-1, RANTES, andother chemokines. It is further contemplated that the upregulation ofcell surface receptors or their ligands such as Fas/Fas ligand, DR4 orDR5/TRAIL would potentiate the apoptotic inducing abilities of thepresent invention by establishment of an autocrine or paracrine effecton hyperproliferative cells. Increased intercellular signaling byelevating the number of GAP junctions would increase theanti-hyperproliferative effects on the neighboring hyperproliferativecell population. In other embodiments, cytostatic or differentiationagents can be used in combination with the present invention to improvethe anti-hyperproliferative efficacy of the treatments. Inhibitors ofcell adhesion are contemplated to improve the efficacy of the presentinvention. Examples of cell adhesion inhibitors are focal adhesionkinase (FAKs) inhibitors and Lovastatin. It is further contemplated thatother agents that increase the sensitivity of a hyperproliferative cellto apoptosis, such as the antibody c225, could be used in combinationwith the present invention to improve the treatment efficacy.

IV. Formulations and Routes for Administration

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions of HDACi and/or hormone therapyagents, or any additional therapeutic agent disclosed herein in a formappropriate for the intended application. Generally, this will entailpreparing compositions that are essentially free of pyrogens, as well asother impurities that could be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention in an effectiveamount may be dissolved or dispersed in a pharmaceutically acceptablecarrier or aqueous medium. Such compositions also are referred to asinocula. The phrase “pharmaceutically or pharmacologically acceptable”refers to molecular entities and compositions that do not produceadverse, allergic, or other untoward reactions when administered to ananimal or a human. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the vectors or cells of the presentinvention, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

The composition(s) of the present invention may be delivered orally,subcutaneously, nasally, intramuscularly, intraperitoneally, orintratumorally. In some embodiments, local or regional delivery of thecomposition thereof, alone or in combination with an additionaltherapeutic agent, to a patient with cancer or pre-cancer conditionswill be a very efficient method of delivery to counteract the clinicaldisease. Similarly, chemo- or radiotherapy may be directed to aparticular, affected region of the subject's body. Regional chemotherapytypically involves targeting anticancer agents to the region of the bodywhere the cancer cells or tumor are located. Other examples of deliveryof the compounds of the present invention that may be employed includeintra-arterial, intracavity, intravesical, intrathecal, intrapleural,and intraperitoneal routes.

Intra-arterial administration is achieved using a catheter that isinserted into an artery to an organ or to an extremity. Typically, apump is attached to the catheter. Intracavity administration describeswhen chemotherapeutic drugs are introduced directly into a body cavitysuch as intravesical (into the bladder), peritoneal (abdominal) cavity,or pleural (chest) cavity. Agents can be given directly via catheter.Intravesical chemotherapy involves a urinary catheter to provide drugsto the bladder, and is thus useful for the treatment of bladder cancer.Intrapleural administration is accomplished using large and small chestcatheters, while a Tenkhoff catheter (a catheter specially designed forremoving or adding large amounts of fluid from or into the peritoneum)or a catheter with an implanted port is used for intraperitonealchemotherapy. Because most drugs do not penetrate the blood/brainbarrier, intrathecal chemotherapy is used to reach cancer cells in thecentral nervous system. To do this, drugs are administered directly intothe cerebrospinal fluid. This method is useful to treat leukemia orcancers that have spread to the spinal cord or brain.

Alternatively, systemic delivery of the chemotherapeutic drugs may beappropriate in certain circumstances, for example, where extensivemetastasis has occurred. Intravenous therapy can be implemented in anumber of ways, such as by peripheral access or through a vascularaccess device (VAD). A VAD is a device that includes a catheter, whichis placed into a large vein in the arm, chest, or neck. It can be usedto administer several drugs simultaneously, for long-term treatment, forcontinuous infusion, and for drugs that are vesicants, which may produceserious injury to skin or muscle. Various types of vascular accessdevices are available.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. This includes but isnot limited to, oral, nasal, or buccal routes. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal or intravenous injection. Suchcompositions would normally be administered as pharmaceuticallyacceptable compositions, described supra. The drugs and agents also maybe administered parenterally or intraperitoneally. The term “parenteral”is generally used to refer to drugs given intravenously,intramuscularly, or subcutaneously.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions also can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The therapeutic compositions of the present invention may beadministered in the form of injectable compositions either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. Thesepreparations also may be emulsified. A typical composition for suchpurpose comprises a pharmaceutically acceptable carrier. For instance,the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg ofhuman serum albumin per milliliter of phosphate buffered saline. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oil and injectable organic esters such asethyloleate. Aqueous carriers include water, alcoholic/aqueoussolutions, saline solutions, parenteral vehicles such as sodiumchloride, Ringer's dextrose, etc. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial agents,anti-oxidants, chelating agents and inert gases. The pH, exactconcentration of the various components, and the pharmaceuticalcomposition are adjusted according to well known parameters. Suitableexcipients for formulation include croscarmellose sodium, hydroxypropylmethylcellulose, iron oxides synthetic), magnesium stearate,microcrystalline cellulose, polyethylene glycol 400, beta-cyclodextran,polysorbate 80, povidone, silicon dioxide, titanium dioxide, and water(purified).

Additional formulations are suitable for oral administration. Oralformulations include such typical excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders. When the route istopical, the form may be a cream, ointment, salve or spray.

V. Kits of the Invention

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, an HDAC inhibitor, an aromatase inhibitor, and/oran anti-androgen may be comprised in a kit. The kits may comprise asuitably aliquoted HDAC inhibitor, an aromatase inhibitor, and/or ananti-androgen, and the components of the kits may be packaged either inaqueous media or in lyophilized form. The container means of the kitswill generally include at least one vial, test tube, flask, bottle,syringe or other container means, into which a component may be placed,and preferably, suitably aliquoted. Where there are more than onecomponent in the kit, the kit also will generally contain a second,third or other additional container into which the additional componentsmay be separately placed. However, various combinations of componentsmay be comprised in a vial. The kits of the present invention also willtypically include a means for containing the HDAC inhibitor, anaromatase inhibitor, and/or an anti-androgen and any other reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow molded plastic containers into which thedesired vials are retained.

Irrespective of the number and/or type of containers, the kits of theinvention may also comprise, and/or be packaged with, an instrument forassisting with the injection/administration and/or placement of acomponent of the kit within the body of an animal or a cell therefrom.Such an instrument may be a syringe, pipette, forceps, and/or any suchmedically approved delivery vehicle, for example.

VI. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Molecular Effects of HDACi on ERA Signaling in ER− BreastCancer Cells Methods:

Cell Viability Assessment using MTT assay:

MTT assay was performed to measure viability of breast cancer cellsafter treatment with various test compounds (Sabnis let al. 2005). IC₅₀and IC₂₅ values for inhibitors is calculated from the linear regressionline of the plot of percentage inhibition versus log inhibitorconcentration. These IC₅₀ values is used for combination or sequencingstudies. The effect of combination or sequence of treatment isdetermined at IC₂₅ of each agent.

Western Immunoblotting for Expression of ERα and Downstream Targets:

The protein extracts from breast cancer (MDA-MB-231 and SKBr3) cellswere subjected to western immunoblotting (Sabnis et al. 2005) to measureprotein expression of ERα following HDACi treatment. Protein expressionof other ER inducible genes such as c-Myc and PgR can also be examined.

Binding Studies:

To confirm affinity of ERα for E₂ and AE tamoxifen, binding studies areperformed (Long et al. 2002). The ligand used for binding studies is₃H-E₂. Receptor saturation curve is plotted before fixing theconcentration of ₃H-E₂ to be used for competitive binding study with4-OHT. The saturation curve is plotted with varying concentration of₃H-E₂ and this enables the inventors to precisely measure the totalnumber of ERαs present inside each cell. The number of receptors iscorrected for total number of cells, since treatment with HDACi may havereduce cell number. The binding affinity of PDs is also tested with ERαin wild type MCF-7 cells.

ER Activity Measurement:

To measure the transcriptional activity of re-expressed ERα in the ER−cells ELISA based ERE activity assay are used (Panomics). This assay isperformed on nuclear extracts of untreated or HDACi pretreated malignantbreast cells. Preparation of nuclear extracts and ERE activity assay isperformed as per manufacture's instructions.

Cell Proliferation in Response to Estrogens:

To examine whether pre-treatment with HDACi, restores mitogenic effectsof estrogens, non-malignant and malignant breast cells are pre-treatedwith HDACi followed by treatment with estradiol (E₂) and the viabilityof cells measured by MTT assay.

Results:

In this study the inventors have utilized ER− MDA-MB-231 cells. Thiscell line exhibits no ERα protein expression by western blotting and arerefractory to growth inhibitory effects of AEs such as tamoxifen andfulvestrant or AIs such as letrozole, exemestane and anastrozole. Inaddition, proliferation of these cells was not affected by E₂. On theother hand, these cells were significantly inhibited by HDACis SAHA andMS-275 as shown in FIG. 1A-FIG. 1B. The IC₅₀ values for SAHA was 205.1nM. The IC₅₀ value for MS-275 was 81.72 nM.

Furthermore, letrozole alone did not inhibit growth of MDA-MB-231 cells,when combined with MS-275 (0.1 nM) and letrozole was found tosynergistically inhibit cell growth in a dose dependent manner(IC₅₀=15.43 nM), as shown in FIG. 1C. FIG. 1D shows that letrozole alonedid not inhibit growth of MDA-MB-231 cells, however, when combined withSAHA, the combination was found to synergistically inhibit cell growthin a dose dependent manner. Similar results were also obtained in cellsthat have acquired resistance to letrozole (LTLT-Ca).

In addition, protein expression of ERα was up-regulated 9.9 and 8 foldafter treatment with HDACis SAHA, MS-275 (10 nM) respectively. Although,HDACi BA was not a potent inhibitor of cell growth (IC₅₀=20.28 mM), ERαprotein expression was up-regulated 15 fold after treatment with 1 μM BAfor 24 hours. This restoration of ERα was also associated withrestoration of response to tamoxifen. The combination of HDACi withtamoxifen was significantly better than single agent alone (p<0.01).

Example 2 Effects on Aromatase Activity

The aromatase activity assay was described by Yue et al. (1997). Theexpression and activation of aromatase was seen after treatment withHDACi. The basal level of aromatase activity in MDA-MB-231 cells wasfound to be 3.02 pmoles/μg of protein/hour. When treated with MS-275 (1μM) for 24 hours and then incubated with 1β-3H-Androstenedione for 18hours, the aromatase activity was found to be 15.193 pmoles/μg ofprotein/hour. This up-regulation of aromatase activity was dosedependent. A similar increase in aromatase activity was observed afterpre-treatment with butyric acid. Also, a 24 hour treatment of MDA-MB-231cells with MS-275 (10 nM), SAHA (10 nM) and BA (1 μM) up-regulated theexpression of aromatase by 2.6, 1.77 and 1.2 fold respectively.

Example 3 In Vivo Dose Response Effects of HDACi

Antitumor efficacies of each HDACi are tested in female mice bearingMDA-MB-231 and SKBr3 tumors using in vivo mouse xenograft model. Thecell lines used for this study are estrogen independent ER− cell lines.SKBr3 cells. These cells are used for studies with AIs, since thesecells have endogenous high levels of aromatase, which is inhibited byAIs. This model simulates advanced and ER−, hormone refractory breastcancer, which is usually associated with mortality of the disease. Thexenograft studies are performed as described by Long et al. 2004;Takabatake et al. 2007. Each agent is given at 5 different doses po andsc and effect on the growth of tumors will be examined. During thecourse of the experiment, tumors are measured weekly with calipers andtumor volume calculated using the formula 4/3πr1²r₂. After completion ofthe treatment the animals are euthanized, tumors and uteri are weighedand collected for further analysis. The weight of the uteri is animportant bioassay for estrogenic/antiestrogenic activity of theadministered agent, since OVX mice have no significant source ofestrogen production. The tumors are examined for expression of signalingproteins in the ER pathway as well as activity of ER and downstreamtargets. The dose of each agent that causes maximum inhibition of tumorgrowth are used for combination studies.

Example 4 Mouse Xenograft Studies Combination of HDACi with AEs/AIs

Experiments are performed to confirm the anti-tumor activity of HDACiand AEs/AIs in mouse xenograft model. The mice receive treatment for aperiod of at least 8 weeks. The groups of mice (n≧10) used in theexperiment are treated with (but may not be limited to) thefollowing: 1. Vehicle treated control, 2. AE alone (sc 5 times a week),3. AI alone (sc 5 times a week), 4. HDACi alone, 5. HDACi plus AE, 6.HDACi plus AI, 8. HDACi and AE in sequence, 9. HDACi and AI in sequence.The dose, frequency and route of administration for HDACi are determinedfrom the above experiments. The dose of AEs and AIs are used asdetermined (Lu et al., 1999) and the drugs will be given sc. During thecourse of experiment, tumor volumes are measured weekly. Afterapproximately 6-8 weeks, the animals are euthanized, tumors and uteriwill be weighed collected for further analysis. Tumor growth rates arecalculated to determine significant effect of the treatment on thetumor. The tumors are examined for expression of signaling proteins inthe ER pathway as well as activity of ER and downstream targets.

Example 5 In Vivo Metastases Mouse Model

To examine the effects of the above-mentioned combinations and sequenceson metastatic spread, a metastasis mouse model is used (Fulton et al.2006; Walser et al. 2006). Here, MDA-MB-231 cells are injectedintravenously into OVX nude mice. All the above-mentioned agents areadministered, and after 4 weeks mice are sacrificed and formation ofpleural metastasis are assessed.

Example 6 HDAC Inhibitors Sensitize ER-Negative Breast Cancer Cells toAromatase Inhibitors

The ability of HDACi to sensitive exemplary ER-negative breast cancercells to aromatase inhibitors was characterized. The following exemplarymaterials and methods were utilized, although one of skill in the artrecognizes that alternative but analogous materials and methods may beemployed.

Cell Line and Cell Proliferation Assay

Exemplary ER negative cell lines (MDA-MB-231) were used. For a cellproliferation assay, 10⁴ cells were plated in a 96 well plate andtreated with indicated drugs for 6 days. The medium was replaced after 3days. On day 7, 500 μg/ml of MTT solution was added to each well andcells incubated for 3 hours. The tetrazolium dye trapped inside themitochondria of the cells was dissolved in DMSO and the absorbance wasmeasured at 560 nm.

Western Immunoblotting

Expression of ER and Aromatase proteins was examined by Westernblotting. β-actin was used as a loading control.

Radiometric ³H2O Release Assay for Aromatase Activity

150,000 cells were plated in IMEM without PR with 5% steroid free serum,1% penicillin-streptomycin and 750 μg/ml G418. Next day the cells wereincubated with 0.5 μCi of [1β³H] androstenedione (Specific activity 23.5Ci/mmole) in 1 ml of media containing 1% charcoal stripped serum for 18hours. The medium was then collected and treated with TCA(trichloroacetic acid) to precipitate proteins. The residual steroids inthe medium were extracted and removed with chloroform and furthertreated with a 2.5% charcoal suspension. The ³H₂O in the supernatant wasmeasured using a scintillation counter. In this assay ³H₂O is releasedduring conversion of [1β³H] Δ4A to estrone, catalyzed by the enzymearomatase. For pre-treatment studies, cells were plated as describedabove and then next day pre-treated with indicated agent for 24 hoursbefore incubating with [1β³H] Δ4A for 18 hours. The activity of theenzyme is corrected for protein concentration in the cells plated andtreated.

Statistics

ANOVA was performed for multiple comparisons. All comparisons are twosided and p of less than 0.05 was considered statistically significant.

The IC₅₀ values of HDAC inhibitors in various cell lines is provided inTable 1 below.

TABLE 1 IC₅₀ values for inhibition of ER- cell proliferation CompoundMDA-MB-231 Hs 578T SKBr3 Her-2 Status − + ++++ SAHA 205.10 nM 16.88 nM80.72 nM MS-275 81.72 nM 28.11 nM 68.2 pM BA 2028 mM 67.91 mM 17.17 μMCI-994 97.79 μM 122.22 nM 8.523 μM

It is demonstrated that the exemplary MDA-MB-231 cells are hormonerefractory and ER-negative, but they have detectable levels of basalaromatase activity (FIG. 2). Also, the growth of the MDA-MB-231 cells isinhibited by HDAC inhibitors (SAHA, MS-275 and BA) in a dose dependentmanner. Furthermore, HDAC inhibitors up-regulate ER and aromataseprotein expression after 24 hour treatment (FIG. 4). The aromataseactivity is upregulated by HDAC inhibitors SAHA, MS-275 and BA in a dosedependent manner (FIG. 3A). FIG. 3B shows that pre-treatment with MS-275increases aromatase activity of MDA-MB-231 cells in a dose-dependentmanner. Finally, when combined with HDACi SAHA or MS-275, letrozoleinhibits the growth of ER-negative MDA-MB-231 cells in a dose dependentmanner with IC₅₀ values of 1.13 μM and 15.43 nM respectively (FIG. 5).

Therefore, histone deacetylase inhibitors can upregulate ER andaromatase protein expression and upregulate aromatase activity andsensitize ER negative breast cancer cells to endocrine therapy. Thecombination of AEs or AIs with HDAC inhibitors represents a new strategyfor the treatment of cancer, including ER-negative breast cancers thatotherwise are treated with chemotherapy.

Example 7 Upregulation of ERA and Aromatase by HDACi MS-275 in an ERNegative Breast Cancer Xenograft Model

The present example concerns the effects of MS-275 (which may also bereferred to as SNDX-275/entinostat) in an exemplary ER negative breastcancer xenograft model. The exemplary cell line MDA-MB-231 was used.

The tumors of MDA-MB-231 cells were grown in female ovariectomized nudemice. Sub-confluent cells were collected into Citric Saline, centrifugedand resuspended in Matrigel (10 mg/mL) at ˜2.5×10⁷ cells/mL. Each mousereceived subcutaneous inoculations in two sites per flank with 100 μL ofcell suspension. Mice were then injected subcutaneously daily with 0.3%HPC in 0.9% NaCl (vehicle) until the tumors reached a measurable size(˜150 mm³). At this point the mice were grouped such that the startingtumor volumes were not statistically different across the groups. Themice were injected with indicated agents (5× weekly) for indicated time.The tumors were measured weekly with calipers and the tumor volumes werecalculated using the formula (4/3)πr₁ ²r₂ (r₁≦r₂).

Expression of ER and aromatase proteins was examined by Westernblotting. β-actin was used as a loading control. The radiometric ³H₂Orelease assay for aromatase activity was performed as follows: thetumors were homogenized using ice-cold DPBS and the homogenate was usedfor the aromatase assay. ³H₂O release assay was performed using [1β³H]androstenedione (Specific activity 23.5 Ci/mmole). The activity of theenzyme is corrected for protein concentration in the tumor homogenates.

ANOVA was performed for multiple comparisons. All comparisons are twosided and p value of less than 0.05 was considered statisticallysignificant.

As shown in FIG. 6, MS-275 inhibits growth of MDA-MB-231 xenografts in adose dependent manner. In FIG. 7, the effect of MS-275 on tumor anduterus weights of the mice. FIGS. 8A-8B provide demonstration thatMS-275 upregulates tumor ER, aromatase expression, and aromataseactivity.

Therefore, the data indicates that MS-275 inhibits growth of MDA-MB-231xenografts and also upregulates expression of ERα, aromatase andandrogen receptor expression in a dose dependent manner. It was alsodetermined that MS-275 is more effective when given orally. MS-275 doesnot affect the uterine weight by itself, indicating no potentialestrogenic or antiestrogenic effects. MS-275 upregulates intra-tumoralaromatase activity. MS-275/SNDX-275/entinostat renders ER negativetumors responsive to endocrine therapy, in particular embodiments of theinvention.

Example 8 Combination Therapy with MS-275 and Letrozole for Cancer

Combination therapy utilizing the exemplary HDAC inhibitor MS-275 andthe exemplary aromatase inhibitor letrozole was investigated. MDA-MB-231xenografts were grown in ovariectomized female nude mice. Mice wereinoculated with 2.5×10⁶ cells per site subcutaneously. When the tumorsreached a measurable size ˜150 mm³, the mice were grouped into 6 groups(n=10), such that the mean tumor volumes across the groups was notstatistically different (p=0.99). The mice were administeredandrostenedione (Δ⁴A) (100 μg/day), Δ4A plus letrozole (10 μg/day),MS-275 (2.5 mg/kg/day), MS-275 plus Δ⁴A, MS-275 plus Δ⁴A plus letrozoleor vehicle. The mice were treated 5 times a week with MS-275 orally andletrozole and Δ⁴A sc. The tumors were measured every week with calipersand the tumor volumes were calculated using the formula, 4/3πr₁ ²r₂.FIG. 9A-9B demonstrate the effect on tumor volume of singular andcombination therapy for MS-275 and letrozole in MDA-MB-231 xenograftmice.

Example 9 Combination Therapy with MS-275 and Letrozole for CellMigration Assay

MDA-MB-231 cells were plated into 60 mm² cell culture dishes and allowedto grow till 80% confluent. The medium was removed and cells were washedwith DPBS twice. The cell monolayer was scraped to make an open partingand the cells were washed twice with DPBS. The cells were then treatedwith indicated agents for 18 hours. The parting was photographed underphase contrast microscope at 0 hour and 18 hour time point. The width ofthe open parting was measured by the on-screen ruler on the microscopecamera. Each parting was measured at 4 locations and the mean distancerecorded (see FIG. 11 for an example). The distance traveled by themigrating cells across the parting at the end of 18 hours was comparedto the initial distance and reported as “width of the open parting”. Themigratory behavior of the cells in this assay may be correlated with theinvasive properties of tumor cells and/or tumor progession. FIG. 10gives an example of the migration of control cells without treatment.FIG. 12 and FIG. 13 gives an example of the migration of cells in thepresence of letrozole or entinostat (MS-275), respectively, and FIG. 14demonstrates the cell migration assay in the presence of the combinationof entinostat and letrozole. The results of the cell migration assay aresummarized in FIGS. 15-16, showing that the combination therapy is moreeffective than either agent alone at limiting cell migration. Thecombination effectively prevented cell migration, and in specificembodiments combinations of HDACi and hormone targeted drugs are usefulfor preventing metastasis of cancer cells.

Example 10 Molecular Effects of HDACi Androgen Negative Prostate CancerCells Methods: Cell Viability Assessment Using MTT Assay:

MTT assay is performed to measure viability of prostate cancer cellsafter treatment with various test compounds (Sabnis let al. 2005). IC50and IC25 values for inhibitors is calculated from the linear regressionline of the plot of percentage inhibition versus log inhibitorconcentration. These IC50 values is used for combination or sequencingstudies. The effect of combination or sequence of treatment is bedetermined at IC25 of each agent.

Western Immunoblotting for Expression of Androgen Receptor andDownstream Targets:

The protein extracts from prostate cancer cells are subjected to westernimmunoblotting (Sabnis et al., 2005). Protein expression of androgenreceptor are examined following HDACi treatment.

Androgen Activity Measurement:

To measure the transcriptional activity of re-expressed androgenreceptor in the androgen receptor negative-cells ELISA based activityassay are used. This assay is performed on nuclear extracts of untreatedor HDACi pretreated malignant prostate cells preparation of nuclearextracts and activity assay is performed as per manufacture'sinstructions (Panomics).

Cell Proliferation in Response to Androgens:

To examine whether pre-treatment with HDACi, restores mitogenic effectsof androgens, non-malignant and malignant prostate cells are pre-treatedwith HDACi followed by treatment with an androgen and the viability ofcells measured by MTT assay.

Example 11 Human Clinical Trial of the Safety and Efficacy ofCombination of HDAC Inhibitor and Aromatase Inhibitor

Objective: To compare the safety and pharmacokinetics of administeredHDAC inhibitor and Aromatase Inhibitor.

Study Design: This will be a Phase I, single-center, open-label,randomized dose escalation study followed by a Phase II study in breastcancer patients with disease that can be biopsied. Patients should nothave had exposure to the HDAC inhibitor or Aromatase Inhibitor prior tothe study entry. Patients must not have received treatment for theircancer within 2 weeks of beginning the trial. Treatments include the useof chemotherapy, hematopoietic growth factors, and biologic therapy suchas monoclonal antibodies. All subjects are evaluated for safety and allblood collections for pharmacokinetic analysis are collected asscheduled. All studies are performed with institutional ethics committeeapproval and patient consent.

Phase I: Patients receive an aromatase inhibitor and HDAC inhibitoraccording to a pre-determined dosing regimen. Cohorts of 3-6 patientsreceive escalating doses of the aromatase inhibitor and the HDACinhibitor until the maximum tolerated dose (MTD) for the combination isdetermined. Test dose ranges are initially determined via theestablished individual dose ranges for the aromatase inhibitor and theHDAC inhibitor. The MTD is defined as the dose preceding that at which 2of 3 or 2 of 6 patients experience dose-limiting toxicity. Dose limitingtoxicities are determined according to the definitions and standards setby the National Cancer Institute (NCI) Common Terminology for AdverseEvents (CTCAE) Version 3.0 (Aug. 9, 2006).

Phase II: Patients receive the aromatase inhibitor as in phase I at theMTD determined in phase I and the HDAC inhibitor as in phase I.Treatment repeats every 6 weeks for 2-6 courses in the absence ofdisease progression or unacceptable toxicity. After completion of 2courses of study therapy, patients who achieve a complete or partialresponse may receive an additional 4 courses. Patients who maintainstable disease for more than 2 months after completion of 6 courses ofstudy therapy may receive an additional 6 courses at the time of diseaseprogression, provided they meet original eligibility criteria.

Blood Sampling: Serial blood is drawn by direct vein puncture before andafter administration of the HDAC inhibitor and/or the HMT inhibitor.Venous blood samples (5 mL) for determination of serum concentrationsare obtained at about 10 minutes prior to dosing and at approximatelythe following times after dosing: days 1, 2, 3, 4, 5, 6, 7, and 14. Eachserum sample is divided into two aliquots. All serum samples are storedat −20° C. Serum samples are shipped on dry ice.

Pharmacokinetics: Patients undergo plasma/serum sample collection forpharmacokinetic evaluation before beginning treatment and at days 1, 2,3, 4, 5, 6, 7, and 14. Pharmacokinetic parameters are calculated bymodel independent methods on a Digital Equipment Corporation VAX 8600computer system using the latest version of the BIOAVL software. Thefollowing pharmacokinetics parameters are determined: peak serumconcentration (Cmax); time to peak serum concentration (tmax); areaunder the concentration-time curve (AUC) from time zero to the lastblood sampling time (AUC0-72) calculated with the use of the lineartrapezoidal rule; and terminal elimination half-life (t1/2), computedfrom the elimination rate constant. The elimination rate constant isestimated by linear regression of consecutive data points in theterminal linear region of the log-linear concentration-time plot. Themean, standard deviation (SD), and coefficient of variation (CV) of thepharmacokinetic parameters are calculated for each treatment. The ratioof the parameter means (preserved formulation/non-preserved formulation)is calculated.

Patient Response to combination therapy: Patient response is assessedvia imaging with X-ray, CT scans, and MRI, and imaging is performedprior to beginning the study and at the end of the first cycle, withadditional imaging performed every four weeks or at the end ofsubsequent cycles. Imaging modalities are chosen based upon the cancertype and feasibility/availability, and the same imaging modality isutilized for similar cancer types as well as throughout each patient'sstudy course. Response rates are determined using the RECIST criteria.(Therasse et al, J. Natl. Cancer Inst. 2000 Feb. 2; 92(3):205-16;http://ctep.cancer.gov/forms/TherasseRECISTJNCI.pdf). Patients alsoundergo cancer/tumor biopsy to assess changes in progenitor cancer cellphenotype and clonogenic growth by flow cytometry, Western blotting, andIHC, and for changes in cytogenetics by FISH. After completion of studytreatment, patients are followed periodically for 4 weeks.

Example 12 Administration of MS-275 and Letrazole for Treatment ofBreast Cancer

According to Example 11, a Human Clinical Trial of the Safety and/orEfficacy of MS-275/letrazole combination therapy is performed. Thebreast cancer patients have not had exposure to either MS-275 orletrazole prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of MS-275 and letrazole will be safe andwell tolerated by cancer patients. The combination of MS-275 andletrazole provides large clinical utility to these cancer patients.

Example 13 Administration of MS-275 and Anastrazole for Treatment ofBreast Cancer

According to Example 11, a Human Clinical Trial of the Safety and/orEfficacy of MS-275/anastrazole combination therapy is performed. Thebreast cancer patients have not had exposure to either MS-275 oranastrazole prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of MS-275 and anastrazole will be safeand well tolerated by cancer patients. The combination of MS-275 andanastrazole provides large clinical utility to these cancer patients.

Example 14 Administration of MS-275 and Exemestane for Treatment ofBreast Cancer

According to Example 11, a Human Clinical Trial of the Safety and/orEfficacy of MS-275/exenestane combination therapy is performed. Thebreast cancer patients have not had exposure to either MS-275 orexemestane prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of MS-275 and exemestane will be safeand well tolerated by cancer patients. The combination of MS-275 andexemestane provides large clinical utility to these cancer patients.

Example 15 Administration of SAHA and Letrazole for Treatment of BreastCancer

According to Example 11, a Human Clinical Trial of the Safety and/orEfficacy of SAHA/letrazole combination therapy is performed. The breastcancer patients have not had exposure to either SAHA or letrazole priorto the study entry and have not received treatment for their cancerwithin 2 weeks of beginning the trial. In conclusion, administration ofa combination of SAHA and letrazole will be safe and well tolerated bycancer patients. The combination of SAHA and letrazole provides largeclinical utility to these cancer patients.

Example 16 Administration of SAHA and Anastrazole for Treatment ofBreast Cancer

According to Example 11, a Human Clinical Trial of the Safety and/orEfficacy of SAHA/anastrazole combination therapy is performed. Thebreast cancer patients have not had exposure to either SAHA oranastrazole prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of SAHA and anastrazole will be safe andwell tolerated by cancer patients. The combination of SAHA andanastrazole provides large clinical utility to these cancer patients.

Example 17 Administration of SAHA and Exemestane for Treatment of BreastCancer

According to Example 11, a Human Clinical Trial of the Safety and/orEfficacy of SAHA/exenestane combination therapy is performed. The breastcancer patients have not had exposure to either SAHA or exemestane priorto the study entry and have not received treatment for their cancerwithin 2 weeks of beginning the trial. In conclusion, administration ofa combination of SAHA and exemestane will be safe and well tolerated bycancer patients. The combination of SAHA and exemestane provides largeclinical utility to these cancer patients.

Example 18 Administration of MGCD-0103 and Letrazole for Treatment ofBreast Cancer

According to Example 11, a Human Clinical Trial of the Safety and/orEfficacy of MS MGCD-0103/letrazole combination therapy is performed. Thebreast cancer patients have not had exposure to either MGCD-0103 orletrazole prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of MGCD-0103 and letrazole will be safeand well tolerated by cancer patients. The combination of MGCD-0103 andletrazole provides large clinical utility to these cancer patients.

Example 19 Administration of MGCD-0103 and Anastrazole for Treatment ofBreast Cancer

According to Example 11, a Human Clinical Trial of the Safety and/orEfficacy of MGCD-0103/anastrazole combination therapy is performed. Thebreast cancer patients have not had exposure to either MGCD-0103 oranastrazole prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of MGCD-0103 and anastrazole will besafe and well tolerated by cancer patients. The combination of MGCD-0103and anastrazole provides large clinical utility to these cancerpatients.

Example 20 Administration of MGCD-0103 and Exemestane for Treatment ofBreast Cancer

According to Example 11, a Human Clinical Trial of the Safety and/orEfficacy of MGCD-0103/exenestane combination therapy is performed. Thebreast cancer patients have not had exposure to either MGCD-0103 orexemestane prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of MGCD-0103 and exemestane will be safeand well tolerated by cancer patients. The combination of MGCD-0103 andexemestane provides large clinical utility to these cancer patients.

Example 21 Administration of FK228 and Letrazole for Treatment of BreastCancer

According to Example 11, a Human Clinical Trial of the Safety and/orEfficacy of FK228/letrazole combination therapy is performed. The breastcancer patients have not had exposure to either FK228 or letrazole priorto the study entry and have not received treatment for their cancerwithin 2 weeks of beginning the trial. In conclusion, administration ofa combination of FK228 and letrazole will be safe and well tolerated bycancer patients. The combination of FK228 and letrazole provides largeclinical utility to these cancer patients.

Example 22 Administration of FK228 and Anastrazole for Treatment ofBreast Cancer

According to Example 11, a Human Clinical Trial of the Safety and/orEfficacy of FK228/anastrazole combination therapy is performed. Thebreast cancer patients have not had exposure to either FK228 oranastrazole prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of FK228 and anastrazole will be safeand well tolerated by cancer patients. The combination of FK228 andanastrazole provides large clinical utility to these cancer patients.

Example 23 Administration of FK-228 and Exemestane for Treatment ofBreast Cancer

According to Example 11, a Human Clinical Trial of the Safety and/orEfficacy of FK228/exenestane combination therapy is performed. Thebreast cancer patients have not had exposure to either FK228 orexemestane prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of FK228 and exemestane will be safe andwell tolerated by cancer patients. The combination of FK228 andexemestane provides large clinical utility to these cancer patients.

Example 24 Administration of LBH589 and Letrazole for Treatment ofBreast Cancer

According to Example 11, a Human Clinical Trial of the Safety and/orEfficacy of LBH589/letrazole combination therapy is performed. Thebreast cancer patients have not had exposure to either LBH589 orletrazole prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of LBH589 and letrazole will be safe andwell tolerated by, cancer patients. The combination of LBH589 andletrazole provides large clinical utility to these cancer patients.

Example 25 Administration of LBH589 and Anastrazole for Treatment ofBreast Cancer

According to Example 11, a Human Clinical Trial of the Safety and/orEfficacy of LBH589/anastrazole combination therapy is performed. Thebreast cancer patients have not had exposure to either LBH589 oranastrazole prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of LBH589 and anastrazole will be safeand well tolerated by cancer patients. The combination of LBH589 andanastrazole provides large clinical utility to these cancer patients.

Example 26 Administration of LBH589 and Exemestane for Treatment ofBreast Cancer

According to Example 11, a Human Clinical Trial of the Safety and/orEfficacy of LBH589/exenestane combination therapy is performed. Thebreast cancer patients have not had exposure to either LBH589 orexemestane prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of LBH589 and exemestane will be safeand well tolerated by cancer patients. The combination of LBH589 andexemestane provides large clinical utility to these cancer patients.

Example 27 Human Clinical Trial of the Safety and Efficacy ofCombination of HDAC Inhibitor and Anti-Androgen

Objective: To compare the safety and pharmacokinetics of administeredHDAC inhibitor and Anti-Androgen.

Study Design: This will be a Phase I, single-center, open-label,randomized dose escalation study followed by a Phase II study inprostate cancer patients with disease that can be biopsied. Patientsshould not have had exposure to the HDAC inhibitor or Anti-Androgenprior to the study entry. Patients must not have received treatment fortheir cancer within 2 weeks of beginning the trial. Treatments includethe use of chemotherapy, hematopoietic growth factors, and biologictherapy such as monoclonal antibodies. All subjects are evaluated forsafety and all blood collections for pharmacokinetic analysis arecollected as scheduled. All studies are performed with institutionalethics committee approval and patient consent.

Phase I: Patients receive an aromatase inhibitor and HDAC inhibitoraccording to a pre-determined dosing regimen. Cohorts of 3-6 patientsreceive escalating doses of the anti-androgen and the HDAC inhibitoruntil the maximum tolerated dose (MTD) for the combination isdetermined. Test dose ranges are initially determined via theestablished individual dose ranges for the anti-androgen and the HDACinhibitor. The MTD is defined as the dose preceding that at which 2 of 3or 2 of 6 patients experience dose-limiting toxicity. Dose limitingtoxicities are determined according to the definitions and standards setby the National Cancer Institute (NCI) Common Terminology for AdverseEvents (CTCAE) Version 3.0 (Aug. 9, 2006).

Phase II: Patients receive the anti-androgen as in phase I at the MTDdetermined in phase I and the HDAC inhibitor as in phase I. Treatmentrepeats every 6 weeks for 2-6 courses in the absence of diseaseprogression or unacceptable toxicity. After completion of 2 courses ofstudy therapy, patients who achieve a complete or partial response mayreceive an additional 4 courses. Patients who maintain stable diseasefor more than 2 months after completion of 6 courses of study therapymay receive an additional 6 courses at the time of disease progression,provided they meet original eligibility criteria.

Blood Sampling: Serial blood is drawn by direct vein puncture before andafter administration of the HDAC inhibitor and/or the HMT inhibitor.Venous blood samples (5 mL) for determination of serum concentrationsare obtained at about 10 minutes prior to dosing and at approximatelythe following times after dosing: days 1, 2, 3, 4, 5, 6, 7, and 14. Eachserum sample is divided into two aliquots. All serum samples are storedat −20° C. Serum samples are shipped on dry ice.

Pharmacokinetics: Patients undergo plasma/serum sample collection forpharmacokinetic evaluation before beginning treatment and at days 1, 2,3, 4, 5, 6, 7, and 14. Pharmacokinetic parameters are calculated bymodel independent methods on a Digital Equipment Corporation VAX 8600computer system using the latest version of the BIOAVL software. Thefollowing pharmacokinetics parameters are determined: peak serumconcentration (Cmax); time to peak serum concentration (tmax); areaunder the concentration-time curve (AUC) from time zero to the lastblood sampling time (AUC0-72) calculated with the use of the lineartrapezoidal rule; and terminal elimination half-life (t1/2), computedfrom the elimination rate constant. The elimination rate constant isestimated by linear regression of consecutive data points in theterminal linear region of the log-linear concentration-time plot. Themean, standard deviation (SD), and coefficient of variation (CV) of thepharmacokinetic parameters are calculated for each treatment. The ratioof the parameter means (preserved formulation/non-preserved formulation)is calculated.

Patient Response to combination therapy: Patient response is assessedvia imaging with X-ray, CT scans, and MRI, and imaging is performedprior to beginning the study and at the end of the first cycle, withadditional imaging performed every four weeks or at the end ofsubsequent cycles. Imaging modalities are chosen based upon the cancertype and feasibility/availability, and the same imaging modality isutilized for similar cancer types as well as throughout each patient'sstudy course. Response rates are determined using the RECIST criteria.(Therasse et al., J. Natl. Cancer Inst. 2000 Feb. 2; 92(3):205-16;http://ctep.cancer.gov/forms/TherasseRECISTJNCI.pdf). Patients alsoundergo cancer/tumor biopsy to assess changes in progenitor cancer cellphenotype and clonogenic growth by flow cytometry, Western blotting, andIHC, and for changes in cytogenetics by FISH. After completion of studytreatment, patients are followed periodically for 4 weeks.

Example 28 Administration of LBH589 and Spironolactone for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of LBH589/spironolactone combination therapy is performed. Theprostate cancer patients have not had exposure to either LBH589 orspironolactone prior to the study entry and have not received treatmentfor their cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of LBH589 and spironolactone will besafe and well tolerated by cancer patients. The combination of LBH589and spironolactone provides large clinical utility to these cancerpatients.

Example 29 Administration of LBH589 and Flutamide for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of LBH589/flutamide combination therapy is performed. Theprostate cancer patients have not had exposure to either LBH589 orflutamide prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of LBH589 and flutamide will be safe andwell tolerated by cancer patients. The combination of LBH589 andflutamide provides large clinical utility to these cancer patients.

Example 30 Administration of LBH589 and Nilutamide for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of LBH589/nilutamide combination therapy is performed. Theprostate cancer patients have not had exposure to either LBH589 ornilutamide prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of LBH589 and nilutamide will be safeand well tolerated by cancer patients. The combination of LBH589 andnilutamide provides large clinical utility to these cancer patients.

Example 31 Administration of LBH589 and Bicalutamide for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of LBH589/bicalutamide combination therapy is performed. Theprostate cancer patients have not had exposure to either LBH589 orbicalutamide prior to the study entry and have not received treatmentfor their cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of LBH589 and bicalutamide will be safeand well tolerated by cancer patients. The combination of LBH589 andbicalutamide provides large clinical utility to these cancer patients.

Example 32 Administration of SAHA and Spironolactone for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of SAHA/spironolactone combination therapy is performed. Theprostate cancer patients have not had exposure to either SAHA orspironolactone prior to the study entry and have not received treatmentfor their cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of SAHA and spironolactone will be safeand well tolerated by cancer patients. The combination of SAHA andspironolactone provides large clinical utility to these cancer patients.

Example 33 Administration of SAHA and Flutamide for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of SAHA/flutamide combination therapy is performed. Theprostate cancer patients have not had exposure to either SAHA orflutamide prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of SAHA and flutamide will be safe andwell tolerated by cancer patients. The combination of SAHA and flutamideprovides large clinical utility to these cancer patients.

Example 34 Administration of SAHA and Nilutamide for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of SAHA/nilutamide combination therapy is performed. Theprostate cancer patients have not had exposure to either SAHA ornilutamide prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of SAHA and nilutamide will be safe andwell tolerated by cancer patients. The combination of SAHA andnilutamide provides large clinical utility to these cancer patients.

Example 35 Administration of SAHA and Bicalutamide for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of SAHA/bicalutamide combination therapy is performed. Theprostate cancer patients have not had exposure to either SAHA orbicalutamide prior to the study entry and have not received treatmentfor their cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of SAHA and bicalutamide will be safeand well tolerated by cancer patients. The combination of SAHA andbicalutamide provides large clinical utility to these cancer patients.

Example 36 Administration of MGCD-0103 and Spironolactone for Treatmentof Prostate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of MGCD-0103/spironolactone combination therapy is performed.The prostate cancer patients have not had exposure to either MGCD-0103or spironolactone prior to the study entry and have not receivedtreatment for their cancer within 2 weeks of beginning the trial. Inconclusion, administration of a combination of MGCD-0103 andspironolactone will be safe and well tolerated by cancer patients. Thecombination of MGCD-0103 and spironolactone provides large clinicalutility to these cancer patients.

Example 37 Administration of MGCD-0103 and Flutamide for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of MGCD-0103/flutamide combination therapy is performed. Theprostate cancer patients have not had exposure to either MGCD-0103 orflutamide prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of MGCD-0103 and flutamide will be safeand well tolerated by cancer patients. The combination of MGCD-0103 andflutamide provides large clinical utility to these cancer patients.

Example 38 Administration of MGCD-0103 and Nilutamide for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of MGCD-0103/nilutamide combination therapy is performed. Theprostate cancer patients have not had exposure to either MGCD-0103 ornilutamide prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of MGCD-0103 and nilutamide will be safeand well tolerated by cancer patients. The combination of MGCD-0103 andnilutamide provides large clinical utility to these cancer patients.

Example 39 Administration of MGCD-0103 and Bicalutamide for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of MGCD-0103/bicalutamide combination therapy is performed. Theprostate cancer patients have not had exposure to either MGCD-0103 orbicalutamide prior to the study entry and have not received treatmentfor their cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of MGCD-0103 and bicalutamide will besafe and well tolerated by cancer patients. The combination of MGCD-0103and bicalutamide provides large clinical utility to these cancerpatients.

Example 40 Administration of MS-275 and Spironolactone for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of MS-275/spironolactone combination therapy is performed. Theprostate cancer patients have not had exposure to either MS-275 orspironolactone prior to the study entry and have not received treatmentfor their cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of MS-275 and spironolactone will besafe and well tolerated by cancer patients. The combination of MS-275and spironolactone provides large clinical utility to these cancerpatients.

Example 41 Administration of MS-275 and Flutamide for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of MS-275/flutamide combination therapy is performed. Theprostate cancer patients have not had exposure to either MS-275 orflutamide prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of MS-275 and flutamide will be safe andwell tolerated by cancer patients. The combination of MS-275 andflutamide provides large clinical utility to these cancer patients.

Example 42 Administration of MS-275 and Nilutamide for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of MS-275/nilutamide combination therapy is performed. Theprostate cancer patients have not had exposure to either MS-275 ornilutamide prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of MS-275 and nilutamide will be safeand well tolerated by cancer patients. The combination of MS-275 andnilutamide provides large clinical utility to these cancer patients.

Example 43 Administration of MS-275 and Bicalutamide for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of MS-275/bicalutamide combination therapy is performed. Theprostate cancer patients have not had exposure to either MS-275 orbicalutamide prior to the study entry and have not received treatmentfor their cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of MS-275 and bicalutamide will be safeand well tolerated by cancer patients. The combination of MS-275 andbicalutamide provides large clinical utility to these cancer patients.

Example 44 Administration of FK228 and Spironolactone for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of FK228/spironolactone combination therapy is performed. Theprostate cancer patients have not had exposure to either FK228 orspironolactone prior to the study entry and have not received treatmentfor their cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of FK228 and spironolactone will be safeand well tolerated by cancer patients. The combination of FK228 andspironolactone provides large clinical utility to these cancer patients.

Example 45 Administration of FK228 and Flutamide for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of FK228/flutamide combination therapy is performed. Theprostate cancer patients have not had exposure to either FK228 orflutamide prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of FK228 and flutamide will be safe andwell tolerated by cancer patients. The combination of FK228 andflutamide provides large clinical utility to these cancer patients.

Example 46 Administration of FK228 and Nilutamide for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of FK228/nilutamide combination therapy is performed. Theprostate cancer patients have not had exposure to either FK228 ornilutamide prior to the study entry and have not received treatment fortheir cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of FK228 and nilutamide will be safe andwell tolerated by cancer patients. The combination of FK228 andnilutamide provides large clinical utility to these cancer patients.

Example 47 Administration of FK228 and Bicalutamide for Treatment ofProstate Cancer

According to Example 27, a Human Clinical Trial of the Safety and/orEfficacy of FK228/bicalutamide combination therapy is performed. Theprostate cancer patients have not had exposure to either FK228 orbicalutamide prior to the study entry and have not received treatmentfor their cancer within 2 weeks of beginning the trial. In conclusion,administration of a combination of FK228 and bicalutamide will be safeand well tolerated by cancer patients. The combination of FK228 andbicalutamide provides large clinical utility to these cancer patients.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

1. A method of treating a hormone resistant cancer in a subject havingor suspected of having the hormone resistant cancer, comprising the stepof administering to the subject an effective amount of the following: ahistone deacetylase inhibitor selected from the group consisting ofPXD-101, LBH589, FK228, MGCD-0103, R306465, PCI-24781, SB-939, ITF-2357,SAHA, CI-994, and MS-275; and an aromatase inhibitor.
 2. The method ofclaim 1, wherein the hormone resistant cancer is breast cancer.
 3. Themethod of claim 1, wherein the aromatase inhibitor is selected from thegroup consisting of anastrozole, exemestane, letrozole, formestane, andtestolactone.
 4. The method of claim 2, wherein the breast cancer isestrogen receptor negative.
 5. The method of claim 2, wherein the breastcancer is estrogen receptor positive.
 6. The method of claim 1, whereinthe histone deacetylase inhibitor and the aromatase inhibitor areadministered simultaneously.
 7. The method of claim 1, wherein thehistone deacetylase inhibitor is administered prior to theadministration of the aromatase inhibitor.
 8. The method of claim 1,wherein the histone deacetylase inhibitor is administered subsequent tothe administration of one or more dose of the aromatase inhibitor. 9.The method of claim 1, wherein the histone deacetylase inhibitor and thearomatase inhibitor act synergistically.
 10. The method of claim 1,wherein the histone deacetylase inhibitor is MS-275.
 11. The method ofclaim 1, wherein the hormone resistance is acquired resistance.
 12. Amethod of treating a hormone resistant cancer in a subject having orsuspected of having the hormone resistant cancer, comprising the step ofadministering to the subject an effective amount of the following: ahistone deacetylase inhibitor; and an aromatase inhibitor selected fromthe group consisting of anastrozole, exemestane, letrozole, formestane,and testolactone.
 13. The method of claim 12, wherein the hormoneresistant cancer is a hormone resistant breast cancer.
 14. The method ofclaim 13, wherein the breast cancer is estrogen receptor negative. 15.The method of claim 13, wherein the breast cancer is estrogen receptorpositive.
 16. The method of claim 12, wherein the histone deacetylaseinhibitor is SAHA, CI-994, PXD-101, LBH589, FK228, MGCD-0103, R306465,PCI-24781, SB-939, ITF-2357, and MS-275.
 17. The method of claim 12,wherein the histone deacetylase inhibitor is MS-275.
 18. The method ofclaim 12, wherein the hormone resistance is acquired resistance.
 19. Amethod of treating a hormone resistant breast cancer in a subject havingor suspected of having the hormone resistant breast cancer, comprisingthe step of administering to the subject an effective amount of thefollowing: a histone deacetylase inhibitor selected from the groupconsisting of SAHA, CI-994, PXD-101, LBH589, FK228, MGCD-0103, R306465,PCI-24781, SB-939, ITF-2357, and MS-275; and an aromatase inhibitorselected from the group consisting of anastrozole, exemestane,letrozole, formestane, testolactone, and a combination thereof.
 20. Themethod of claim 19, wherein the breast cancer is ER−, PR+.
 21. Themethod of claim 19, wherein the breast cancer is ER−, PR− and Her2+. 22.The method of claim 19, wherein the breast cancer is ER−, PR− and Her2−.23. The method of claim 19, wherein the histone deacetylase inhibitor isMS-275.
 24. The method of claim 19, wherein the hormone resistance isacquired resistance.
 25. A method of inhibiting growth of a cellcomprising contacting the cell with an aromatase inhibitor and a histonedeacetylase inhibitor, each in an effective amount to inhibit growth ofsaid cell, wherein the histone deacetylase inhibitor is selected fromthe group consisting of PXD-101, LBH589, FK228, MGCD-0103, R306465,PCI-24781, SB-939, ITF-2357, SAHA and CI-994 and MS-275.
 26. The methodof claim 25, wherein the aromatase inhibitor is selected from the groupconsisting of anastrozole, exemestane, letrozole, formestane,testolactone, and a combination thereof.
 27. The method of claim 25,wherein the histone deacetylase inhibitor is MS-275.
 28. The method ofclaim 25, wherein the hormone resistance is acquired resistance.
 29. Amethod of inhibiting growth of a cell comprising contacting the cellwith an aromatase inhibitor and a histone deacetylase inhibitor, each inan effective amount to inhibit growth of said cell, wherein thearomatase inhibitor is selected from the group consisting ofanastrozole, exemestane, letrozole, formestane, testolactone, and acombination thereof.
 30. The method of claim 29, wherein the cell is arefractory cancer cell and the hormone resistance is acquiredresistance.
 31. The methods of claim 29, wherein the histone deacetylaseinhibitor increases the expression of an estrogen receptor on the cell.32. The method of claim 29, wherein the histone deacetylase inhibitorincreases the expression or activity of aromatase in the cell.
 33. Themethod of claim 29, wherein the contacting occurs in vivo.
 34. Themethod of claim 29, wherein the histone deacetylase inhibitor is MS-275.35. The method of claim 29, wherein the hormone resistance is acquiredresistance.
 36. A method of treating a hormone resistant cancer in asubject having or suspected of having the hormone resistant cancer,comprising the step of administering to the subject an effective amountof the following: a histone deacetylase inhibitor; and an anti-androgen,wherein the anti-androgen is not a 17alpha-hydroxylase-C17,20-lyaseinhibitor or a 5-reductase inhibitor.
 37. The method of claim 36,wherein the hormone resistant cancer is prostate cancer.
 38. The methodof claim 36, wherein the histone deacetylase inhibitor is a selectivehistone deacetylase inhibitor.
 39. The method of claim 36, wherein thehistone deacetylase inhibitor is selected from the group consisting ofSAHA, CI-994, PXD-101, LBH589, FK228, MGCD-0103, R306465, PCI-24781,SB-939, ITF-2357, and MS-275.
 40. The method of claim 36, wherein theanti-androgen is selected from the group consisting of spironolactone(Aldactone, Spiritone; Novo-Spiroton, Spiractin, Verospiron orBerlactone), cyproterone acetate (Androcur, Climen, Diane 35, Ginette35), flutamide (Eulexin), nilutamide (Anandron, Nilandron), bicalutamide(Casodex) and a combination thereof.
 41. The method of claim 36, whereinthe histone deacetylase inhibitor and the anti-androgen are administeredsimultaneously.
 42. The method of claim 36, wherein the histonedeacetylase inhibitor is administered prior to the administration of theanti-androgen.
 43. The method of claim 36, wherein the histonedeacetylase inhibitor and the anti-androgen act synergistically.
 44. Themethod of claim 36, wherein the hormone resistance is acquiredresistance.